Proteins toxic to Hemipteran insect species

ABSTRACT

The present invention discloses Hemipteran insect inhibitory proteins, methods of using such proteins, nucleotide sequences encoding such proteins, methods of detecting and isolating such proteins, and their use in agricultural systems.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/015,957, filed Feb. 4, 2016, which is a continuation of U.S.application Ser. No. 13/857,196, filed Apr. 5, 2013, which claimspriority to U.S. Provisional Application Ser. No. 61/621,436, filed Apr.6, 2012, all of which are incorporated herein by reference in itsentirety their entireties.

INCORPORATION OF SEQUENCE LISTING

The Sequence Listing contained in the file named “P34307US04.txt”, whichis 529,878 bytes in size (measured in operating system MS-Windows) andwas created on Jul. 17, 2017, is contemporaneously filed by electronicsubmission (using the United States Patent Office EFS-Web filing system)and is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to the field of insectinhibitory proteins. In particular, the present invention relates toproteins exhibiting insect inhibitory activity against agriculturallyrelevant pests of crop plants and seeds, particularly Hemipteran speciesof insect pests.

BACKGROUND OF THE INVENTION

Insect inhibitory proteins derived from Bacillus thuringiensis (Bt) arenon-toxic to humans, vertebrates, and plants. These proteins are alsobiodegradable, safe, and effective in controlling pest insects. Some ofthese proteins have been and are being used to control agriculturallyrelevant pests of crop plants by spraying plants with formulationscontaining these proteins or with microorganisms that express them,treating seeds with treatments containing these proteins, or expressingthese proteins in crop plants and seeds of crop plants asplant-incorporated protectants.

Certain Hemiptera species, particularly Amrasca, Empoasca and Lygusbugs, are pests of cotton and alfalfa, and typically are only controlledusing broad spectrum chemistries, e.g., endosulfan, acephate, andoxamyl, which can persist in and are harmful to the environment. A fewBt proteins have been developed in formulations or as transgenic traitsin crop plants for commercial use by farmers to control Coleopteran andLepidopteran pest species, but no Bt proteins have been developed foruse in commercial control of Hemipteran pest species.

Hemipteran specific toxic proteins have been reported in the art. TIC807is a Bacillus thuringiensis protein disclosed in U.S. Patent ApplicationPublication No. US 2008-0295207 A1 as being toxic to Hemipteran pestspecies. A Cry51Aa1 protein reported as toxic to Lepidopteran speciesthat closely resembles the amino acid sequence of TIC807 has also beendisclosed (Huang et al., (2007) J. Invertebr. Pathol. 95(3), 175-180),but no Hemipteran specific activity was reported. Baum et al. disclosedTIC853, a protein reported to be toxic to Lygus pest species (U.S.Patent Application Publication No. US 2010-0064394 A1). A proteinreferred to as AXMI-171 was reported to exhibit some limited inhibitionof Hemipteran insects (U.S. Patent Application Publication No.US2010-0298207 A1, example 18), particularly Lygus hesperus.

All of these proteins exhibit a narrow range of toxicity only againstLygus hesperus and exhibit toxic effects against other Lygus pestspecies only in high doses which are not considered to be achievable byexpression in plants. Compared to the Hemipteran toxic proteins in theprior art, there is a need for toxin proteins that can be used on and inplants that exhibit a broad host range against Hemipteran pest speciesand at low concentration effective doses.

BRIEF SUMMARY OF THE INVENTION

Recombinantly engineered Hemipteran toxic proteins described herein(referred to herein as “engineered toxin proteins”, “engineered toxicproteins”, “engineered Hemipteran toxic proteins”, or “engineeredHemipteran toxin proteins”, are also referred to herein in truncatedform as “eHTP's” when referred to in groups of two or more suchproteins, and “eHTP” when referred to singularly) are derivatives ofnaturally occurring Bacillus thuringiensis insecticidal toxins, TIC807(SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ ID NO:182), TIC853(SEQ ID NO:184), and AXMI-171 (SEQ ID NO:206) have been describedpreviously to exhibit bio-control activity directed to Hemipteran pestspecies, particularly Lygus hesperus insect species (references citedelsewhere herein). The recombinant Hemipteran insect toxic proteins ofthe present invention are particularly toxic to insects of the Amrasca,Empoasca and Lygus species of insect pests and to other insect pestspecies that are phylogenetically related to each of these species ofinsect pests, and additionally to insect pests that feed on plants usinga piercing and sucking mechanism used by the pest species Amrasca,Empoasca and Lygus species of the order Hemiptera. Unlike the precursorinsecticidal toxins TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8),Cry51Aa1 (SEQ ID NO:182), TIC853 (SEQ ID NO:184), and AXMI-171 (SEQ IDNO:206) from which they are derived, which each require moderately highto high doses of protein to achieve toxic effects upon one Lygus speciesand exhibit very low or virtually undetectable toxic effects upon asecond closely related species of Lygus, the eHTP proteins of thepresent invention exhibit surprising and unexpected low dose toxiceffects against insect pests of the order Hemiptera, including hostrange toxic effects that span the spectrum of pests within the order.

The eHTP's of the present invention each contain at least one amino acidsubstitution, one amino acid addition, or one amino acid deletioncompared to the primary amino acid sequence of one or more of the toxinproteins set forth in any of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:182, orSEQ ID NO:184. In certain embodiments, an eHTP is provided that containsat least from about 2 to about 260 fold greater inhibitory activityagainst a Lygus pest species than any one or more of the toxins setforth in any of SEQ ID NO:2 (TIC807), SEQ ID NO:8 (TIC807_M2), SEQ IDNO:182 (Cry51Aa1), SEQ ID NO:184 (TIC853), and/or SEQ ID NO:206(AXMI-171). Optionally the eHTP exhibits at least about 95% amino acidsequence identity to the toxin protein selected from the groupconsisting of SEQ ID NO:2 (TIC807) and SEQ ID NO:182 (Cry51Aa1). Incertain embodiments, an eHTP is provided that contains at least oneamino acid substitution, at least one amino acid addition, or at leastone amino acid deletion when compared to the amino acid sequence of anyof SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:182, or SEQ ID NO:184. The eHTPexhibits an increased or greater Lygus inhibitory activity and targetpest species spectrum compared to the activity and target pest speciesspectrum of the Bacillus thuringiensis proteins of set forth in SEQ IDNO:2, SEQ ID NO:8, SEQ ID NO:182, and SEQ ID NO:184. Each of theaforementioned eHTP's contain at least, collectively or in thealternative: (i) the amino acid substitution, addition, or deletion in asolvent accessible amino acid residue of SEQ ID NO:2; (ii) the aminoacid substitution, addition, or deletion within 3 consecutive residuesof a solvent accessible amino acid residue of SEQ ID NO:2; or, (iii) anamino acid sequence as set forth in SEQ ID NO:180. The aforementionedeHTP's will each contain at least, with reference to the amino acidsequence positions as numbered according to the amino acid positions ofTIC807, one substitution or deletion selected from the group consistingof asparagine at position 12 replaced by aspartic acid, phenylalanine atposition 46 replaced by serine, isoleucine at position 52 replaced bymethionine, tyrosine at position 54 replaced by histidine, threonine atposition 68 replaced by alanine, glutamine at position 70 replaced byalanine, alanine at position 87 replaced by serine, threonine atposition 93 replaced by alanine, serine at position 95 replaced byalanine, glycines at position 105 replaced by alanine, serine atposition 117 replaced by alanine, serine at position 119 replaced byalanine, glutamate at position 125 replaced by cysteine, histidine,arginine, phenylalanine, serine, glutamine, lysine, threonine,asparagine, alanine, leucine, valine, methionine, aspartic acid, ortyrosine, glycines at position 128 replaced by alanine, threonine atposition 133 replaced by glutamic acid, tyrosine, or tryptophan,isoleucine at position 134 replaced by alanine, valine, leucine,phenylalanine, lysine, cysteine, or methionine, glutamate at position135 replaced by serine, alanine, valine, tryptophan, or threonine,asparagine at position 137 replaced by histidine, tyrosine, threonine,glutamic acid, serine, alanine, glutamine, glycine, isoleucine,tryptophan, lysine, cysteine, methionine, aspartic acid, phenylalanine,or arginine, phenylalanine at position 138 replaced by valine, Ala139replaced by serine, Thr145 replaced by alanine, Phe147 replaced byserine, valine, threonine, cysteine, leucine, aspartic acid, alanine,glycine, glutamic acid, isoleucine, tyrosine, methionine, asparagine,glutamine, hystidine, alanine, arginine, tryptophan, or proline,glutamine at position 148 replaced by alanine, glutamine at position 149replaced by aspartic acid, glutamic acid, cysteine, alanine, orphenylalanine, alanine at position 150 replaced by serine, leucine,valine, glycine, aspartic acid, tryptophan, glutamic acid, asparagine,tyrosine, phenylalanine, proline, lysine, threonine, glutamine, orarginine, seroine at position 151 replaced by alanine, aspartate atposition 153 replaced by alanine, glutamate at position 155 replaced bycysteine, isoleucine, lysine, aspartic acid, histidine, tyrosine,glutamine, lysine, asparagine, threonine, alanine, phenylalanine,arginine, methionine, proline, tryptophan, serine, or valine, asparagineat position 157 replaced by cysteine, aspartic acid, tryptophan,tyrosine, methionine, alanine, phenylalanine, valine, leucine, proline,glutamic acid, threonine, glycine, isoleucine, or arginine, isoleucineat position 158 replaced by alanine, serine at position 159 replaced byalanine or threonine, serine at position 167 replaced by arginine oralanine, valine at position 175 replaced by alanine, methionine atposition 177 replaced by alanine, asparagine at position 180 replaced byaspartic acid, threonine at position 182 replaced by alanine, leucine atposition 187 replaced by alanine, histidine at position 196 deleted,tyrosine at position 197 deleted, serine at position 198 deleted,histidine at position 199 deleted, tyrosine at position 200 replaced byalanine, tyrosine at position 200 deleted, Ser201 replaced by alanine,serine at position 201 deletion, tryptophan at position 208 replaced byalanine, serine at position 217 replaced by asparagine, proline atposition 219 replaced by arginine, tryptophan at position 223 replacedby tyrosine, phenylalanine at position 235 replaced by alanine,asparagine at position 239 replaced by alanine, aspartate at position241 replaced by alanine, threonine at position 243 replaced by alanine,valine at position 244 replaced by isoleucine, threonine at position 245replaced by alanine, tyrosine at position 246 replaced by phenylalanine,threonine at position 247 replaced by alanine or lysine, serine atposition 249 replaced by alanine or arginine, valine at position 250replaced by alanine, valine at position 251 replaced by alanine, serineat position 252 replaced by alanine, arginine at position 273 replacedby tryptophan, threonine at position 274 replaced by alanine, isoleucineat position 275 replaced by alanine, arginine at position 282 replacedby alanine, histidine at position 287 replaced by alanine orphenylalanine, serine at position 293 replaced by alanine, asparagine atposition 295 replaced by alanine, glutamate at position 299 replaced byalanine, methionine at position 300 replaced by alanine, threonine atposition 303 replaced by alanine, proline at position 305 replaced byalanine, isoleucine at position 306 replaced by alanine, and threonineat position 308 replaced by alanine, or wherein the protein comprisesany combination of the referenced substitutions and/or deletions. eHTP'scontain at least one amino acid substitution, one amino acid addition,or one amino acid deletion at an amino acid residue of SEQ ID NO:2, orthe corresponding amino acid position of SEQ ID NO:8, SEQ ID NO:182, orSEQ ID NO:184, selected from the group consisting of (i) an amino acidresidue having a relative solvent-accessibility of from at least about15% to at least about 36%; and (ii) an amino acid residue located withina distance of about 3 consecutive residues from an amino acid havingfrom at least about 15% to at least about 36% relativesolvent-accessibility. An eHTP of the present invention contains atleast one amino acid substitution, addition, or deletion at an aminoacid residue selected from the group consisting of Thr93, Ser95, Ser97,Phe147, Gln149, Ser151, Asn180, Thr182, Val251, Gln253, and Ser255 ofSEQ ID NO:2. Any of the aforementioned eHTP's can contain at least oneadditional amino acid substitution, addition, or deletion at an aminoacid residue selected from the group consisting of Val10, Ile14, Asn22,Asn23, Gly24, Ile25, Gln26, Gly27, Phe30, Gln38, Ile39, Asp40, Thr41,Ile43, Ser193, Thr194, Glu195, His196, Tyr197, Ser198, His199, Tyr200,Ser201, Gly202, Tyr203, Pro204, Ile205, Leu206, Thr207, Trp208, Ile209,Ser210, Tyr216, Ser217, Gly218, Pro219, Pro220, Met221, Ser222, Trp223,Tyr224, Phe225, Asn239, and Val244 of SEQ ID NO: 2 or the correspondingamino acid residue position of SEQ ID NO:8, SEQ ID NO:182, or SEQ IDNO:184. Any of the aforementioned eHTP's may contain one or moremodifications selected from the group consisting of S95A, F147A, Q149E,V251A, P219R, and a deletion of any three consecutive amino acids fromamino acid residues 196-201 as set forth in SEQ ID NO:2. Any of theeHTP's of the present invention can be further modified to exhibitincreased solubility compared to the underlying naturally occurringBacillus thuringiensis protein as set forth in any of SEQ ID NO:2, SEQID NO:8, SEQ ID NO:182, or SEQ ID NO:184 in which the eHTP contains atleast one or more amino acid sequence modifications relative to theamino acid sequence as set forth in SEQ ID NO:2. The modification(s)contain at least a lysine substitution at one or more of the amino acidpositions defined as 58, 59, 198, 199, 201, or 202 in SEQ ID NO:2; aglutamic acid residue substitution at one or more of the amino acidpositions defined as 198, 248, or 301 in SEQ ID NO:2; or an arginineresidue substitution at one or more of the amino acid positions definedas 246, 250, or 253 in SEQ ID NO:2. An eHTP having an amino acidsequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65,SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70,SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75,SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80,SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85,SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90,SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100,SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ IDNO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114,SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ IDNO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:13, SEQID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128,SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ IDNO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142,SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ IDNO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156,SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ IDNO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170,SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ IDNO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQID NO:202, and SEQ ID NO:204, or an insect inhibitory fragment thereof,is a preferred embodiment of the present invention. The targetHemipteran pest species inhibited by the eHTP's of the present inventioninclude at least Lygus hesperus, Lygus lineolaris, Empoasca fabae andAmrasca devastans, as well as other pests within the order Hemipterathat are phylogenetically related to each other or which use a piercingand sucking approach for feeding on plants.

Methods of controlling a Hemipteran pest by contacting the pest with aHemipteran inhibitory amount of a eHTP of the present invention, as wellas an insect inhibitory composition that contains at least a Hemipterancontrolling amount (or Hemipteran inhibitory amount) of one or more ofthe eHTP's of the present invention, are also provided. In certainembodiments, an insect inhibitory composition comprising any of theeHTP's disclosed herein is provided. In certain embodiments of thesemethods, the Hemipteran pest is in a cotton field, a soybean field or analfalfa field. Hemipteran toxic or Hemipteran controlling compositionscan contain at least one or more eHTP along with a supplemental agentthat is selected from the group consisting of an insect inhibitoryprotein, an insect inhibitory dsRNA molecule, and an insect inhibitorychemistry. Each of these agents can exhibit Hemipteran controllingproperties, can exhibit properties for controlling pests unrelated toHemipteran species such as Lepidopteran species or Coleopteran species,or may exhibit dual mode of action properties in which one or moreHemipteran species and one or more Lepidopteran or Coleopteran speciesare simultaneously controlled.

Recombinant polynucleotides that encode eHTP's of the present inventionare provided. Microbes are also provided that contain thepolynucleotides of the present invention, and such polynucleotideswithin such microbes are functionally positioned within expressioncassettes designed to express the eHTP's of the present invention fromoperably linked functional genetic regulatory elements. Microbes areintended to include bacterial cells, as well as transgenic plant cells.Such transgenic plant cells can be regenerated into whole plants, orplant parts that also contain the recombinant polynucleotide. Methods ofcontrolling a Hemipteran pest by exposing the pest to the microbe,whether bacterial cell or transgenic plant cell, plant or plant part,each of which expresses a Hemipteran inhibitory amount of an eHTP arealso provided. The recombinant polynucleotide may contain a nucleotidesequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194,SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ IDNO:199, SEQ ID NO:200, SEQ ID NO:201, and SEQ ID NO:203, or othersequences that can be assembled to encode one or more of the proteins ofthe present invention. In certain embodiments, the recombinantpolynucleotide can further comprise a nucleotide sequence encoding oneor more insect inhibitory agents that are different from the eHTPencoded by the recombinant polynucleotide. The transgenic plant part isa seed, a boll, a leaf, a flower, pollen, a stem, a root, or any portionthereof. The transgenic plant part may be a non-regenerable portion ofthe seed, boll, leaf, flower, stem, or root. Also provided are methodsof controlling a Hemipteran pest, comprising exposing the transgenicmicrobe, bacteria, plant cell, plant or plant part to the target pest,wherein the microbe, bacteria, plant cell, plant or plant part expressesa Hemipteran inhibitory amount of a eHTP encoded by the recombinantpolynucleotide.

Processed plant products that contain a detectable amount of arecombinant polynucleotide encoding an eHTP or any distinguishingHemipteran controlling portion thereof are also provided. Such processedproducts include, but are not limited to, plant biomass, oil, meal,animal feed, flour, flakes, bran, lint, hulls, and processed seed. Theprocessed product may be non-regenerable.

Methods of making a transgenic plant by introducing the recombinantpolynucleotide into a plant cell and selecting a transgenic plant thatexpresses an insect inhibitory amount of an eHTP encoded by arecombinant polynucleotide are also provided. The methods includeintroducing the recombinant polynucleotide encoding any of the eHTP'sprovided herein into a plant cell and selecting a transgenic plant thatexpresses an insect inhibitory amount of the eHTP encoded by therecombinant polynucleotide.

Other embodiments, features, and advantages of the invention will beapparent from the following detailed description, the examples, and theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the mortality of Lygus species plotted against eHTPprotein concentration. FIG. 1A illustrates the mortality of Lygushesperus populations in response to various concentrations of fourdifferent eHTP's compared to a control sample containing the naturallyoccurring TIC807 protein. FIG. 1B illustrates the mortality of Lyguslineolaris populations in response to various protein concentrations ofthree different eHTP's compared to a control sample containing thenaturally occurring TIC807 protein.

FIG. 2 illustrates a ribbon diagram of the atomic structure of aHemipteran toxic protein of the present invention showing the relativepositions of the result effective changes increasing toxic effectsand/or broadening host range specificity compared to the relativeposition of the same amino acid position within a TIC807 or relatedprotein. Two surface patches are illustrated by spheres encirclingparticular residue positions within the atomic structure in the ribbondiagram: [1] one sphere has an atomic radius of from about 9.2 to about12.2 Angstroms from the beta carbon atom of S95 (relative to the S95position as set forth in SEQ ID NO:2); [2] another sphere has an atomicradius of from about 9.2 to about 12.2 Angstroms from the beta carbonatom of P219 (relative to the P219 position as set forth in SEQ IDNO:2). Changes to the amino acids within the ribbon structure that fallwithin these spheres are result effective in causing increased toxicproperties and broader host range toxic effects compared to a proteinhaving a naturally occurring amino acid at that particular position.

FIG. 3 is a chart view illustrating the population mortality of Lygusspecies for thirteen different eHTP's compared to each other and to thenaturally occurring TIC807 protein.

DETAILED DESCRIPTION

This application describes eHTP's (engineered Hemipteran species toxicproteins). The eHTP's of the present invention are to be distinguishedfrom proteins such as TIC807, TIC853, Cry51Aa1 and AXMI-171, which areknown in the art and are not to be considered to be within the scope ordefinition of the term eHTP, as the prior art proteins are notengineered to exhibit improved toxic properties directed to one or moreHemipteran pest species and do not exhibit broad host range levels ofinhibitory activity. eHTP's surprisingly and unexpectedly exhibit highlevels of toxic activity against Hemipteran and related pest species. Anadditional feature of these eHTP's that is even more unexpected andsurprising is the finding that these proteins exhibit broader host rangetoxic properties compared to progenitor proteins which provide thefoundational basis for the eHTP's of the present invention. Thefoundational or baseline scaffold toxin proteins, such as TIC807 (SEQ IDNO:2), Cry51Aa1 (SEQ ID NO:8), TIC853 (SEQ ID NO:184), and AXMI-171 (SEQID NO:206) do not exhibit the breadth and scope of biologicalanti-Hemipteran activity or host range of the eHTP proteins of thepresent invention.

More than 2000 different amino acid sequence variants of Hemipterantoxic proteins derived from Bacillus thuringiensis species were testedto identify the specific amino acid insertions, substitutions, ordeletions described herein which confer expanded Hemipteran species hostrange inhibitory spectrum and also provide dramatically increasedHemipteran species inhibitory activity when compared to the spectrum andactivity of the baseline scaffold protein, TIC807, TIC853, and Cry51Aa1.Amino acid residues are identified in the baseline scaffold proteinsthat (a) can be modified to yield enhanced Hemipteran inhibitoryspectrum and/or improved Lygus inhibitory activity relative to one ormore of the scaffold proteins, (b) accumulate in surface patches of afolded insect inhibitory protein exhibiting the fold structure of one ormore of the scaffold proteins, and/or (c) occur in specific positions ofone or more of the scaffold protein amino acid sequence that are resulteffective in decreasing the resulting eHTP proteins' mean effective dosefor controlling Hemipteran species and broadening the range ofHemipteran species that are affected by the eHTP protein.

The Hemipteran pest species are intended to mean insects that feed uponplants and plant tissues by slashing or piercing the outer surface ofthe target plant, and then consume macerated plant exudates pooling inthe slash or pierce location by sucking or wicking the pooled exudates.Such insects include adults and nymphs, including but not limited to thefollowing listing of plant bugs: the Family Miridae, cicadas from theFamily Cicadidae, leafhoppers (e. g., Empoasca spp., Amrasca spp.) fromthe Family Cicadellidae, planthoppers from the families Fulgoroidea andDelphacidae, treehoppers from the Family Membracidae, psyllids from theFamily Psyllidae, whiteflies from the Family Aleyrodidae, aphids fromthe Family Aphididae, phylloxera from the Family Phylloxeridae,mealybugs from the Family Pseudococcidae, scales from the familiesCoccidae, Diaspididae and Margarodidae, lace bugs from the FamilyTingidae, stink bugs from the Family Pentatomidae, cinch bugs (e. g.,Blissus spp.) and other seed bugs from the Family Lygaeidae, spittlebugsfrom the Family Cercopidae squash bugs from the Family Coreidae, and redbugs and cotton stainers from the Family Pyrrhocoridae. Other pests fromthe order Hemiptera include Acrosternum hilare (green stink bug), Anasatristis (squash bug), Blissus leucopterus leucopterus (chinch bug),Corythuca gossypii (cotton lace bug), Cyrtopeltis modesta (tomato bug),Dysdercus suturellus (cotton stainer), Euschistus serous (brown stinkbug), Euschistus variolarius (one-spotted stink bug), Graptostethus spp.(complex of seed bugs), Leptoglossus corculus (leaf-footed pine seedbug), Lygus lineolaris (tarnished plant bug), Lygus hesperus (Westerntarnish plant bug), Nezara viridula (southern green stink bug), Oebaluspugnax (rice stink bug), Oncopeltus fasciatus (large milkweed bug), andPseudatomoscelis seriatus (cotton fleahopper). More specifically, theFamily Cicadellidae includes, but is not limited to the tribeEmpoascini, e.g. Amrasca biguttula, Amrasca devastans, Austroascaviridigrisea, Asymmetrasca decedens, Empoasca decipiens, Empoascadistinguenda, Empoasca dolichi, Empoasca fabae, Empoasca kerri, Empoascakraemeri, Empoasca onukii, Empoasca sakaii, Empoasca smithi, Empoascavitis, Jacobiasca lybica, Sonasasca solana, tribe Erythroneurini, e.g.Empoascanara nagpurensis, Thaiaassamensis, Zygnidia quyumi, tribeNirvaniae, e.g. Sophonia rufofascia, Family Delphacidae, e.g.Nilapoarvata lugens, Sogatella furcifera, Unkanodes sapporonus, andFamily Lophopidae, e.g. Zophiuma lobulata.

eHTP's of the present invention contain one or more amino acid sequencemodifications compared to one or more of the scaffold proteins,including substitutions and deletions, of amino acid residues atseventy-two (72) different amino acid positions. Such modificationsprovide eHTP's with increased toxicity and/or an enhanced inhibitoryspectrum against Hemipteran insects when compared to one or more of thescaffold proteins which include but are not limited to TIC807 (SEQ IDNO:2), or related protein such as TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQID NO:182), and TIC853 (SEQ ID NO:184). eHTP's include, but are notlimited to, modifications of at least one amino acid substitution or oneamino acid deletion at any of these seventy-two positions, described as“X” in the amino acid sequence set forth as SEQ ID NO:180 but do notinclude the amino acid sequences of SEQ ID NO:2, SEQ ID NO:8, SEQ IDNO:182, or SEQ ID NO:184. eHTP's of the present invention also exhibitenhanced Hemipteran inhibitory spectrum and/or improved Hemipteraninhibitory activity when compared to the spectrum and activity of thebaseline or scaffold proteins.

eHTP's include at least one amino acid modification of the relativepositions of TIC807 (SEQ ID NO:2). eHTP's can also include at least two,three, four, or more of these aforementioned amino acid substitutionsand/or deletions and can also include at least two, three, four, or moreof these amino acid substitutions and/or deletions as well as a deletionof any three contiguous amino acids within residues 196-201 of SEQ IDNO:2. Accordingly, eHTP's include proteins set forth as SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ IDNO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109,SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ IDNO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123,SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ IDNO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137,SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ IDNO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151,SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ IDNO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165,SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ IDNO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179,SEQ ID NO:202, and SEQ ID NO:204, and insect inhibitory fragmentsthereof.

eHTP's of the present invention exhibit any amino acid sequencedifferent from any one or more of the scaffold proteins, including SEQID NO:2 (TIC807), in at least one amino acid position where thedifferent amino acid residue either (i) has a relative amino acidsolvent-accessibility of at least from about 15% to at least about 36%compared to the same residue positions in any one or more of thescaffold proteins; and/or (ii) is located within a distance of about 3consecutive amino acid residues from an amino acid having at least fromabout 15% to at least about 36% relative solvent-accessibility comparedto the corresponding amino acid residue positions in the primary aminoacid sequence of one or more of the scaffold proteins, and exhibitsbroadened Hemipteran inhibitory spectrum and/or increased Hemipteraninhibitory activity when compared to the activity correlated with one ormore of the scaffold proteins. The words “increased spectrum” areintended to mean, with reference to two different proteins exhibitingtoxic effects to a particular single pest, the protein exhibitingincreased spectrum exhibits toxic effects to that particular single pestas well as to one or more other pests within the same phylogenetic orderor to one or more other pests in one or more different phylogeneticorders other than the order to which the particular single pest belongs.The words “increased Hemipteran inhibitory activity” are intended tomean that a particular protein exhibiting such increased activityrequires, under standardized conditions, a lower amount of that proteinto achieve a particular affect, such as mortality, stunting, morbidity,cessation of feeding, or another measureable phenotypic effect upon aparticular single pest, than a control protein.

eHTP's exhibit an amino acid sequence that differs from one or more ofthe scaffold proteins, including particularly TIC807, in at least oneamino acid residue located within at least one of the two differentsurface patches of a folded insect inhibitory protein (see FIG. 2 andTable 3 data). One surface patch is defined as including the amino acidresidues encompassed within a sphere having an atomic radius of fromabout 9.2 to about 12.2 Angstroms (FIG. 2, sphere [1]) relative to thebeta-carbon (Cb) atom of Ser95 as set forth in SEQ ID NO:2 when thatprotein is folded into a three dimensional structure under physiologicalconditions; which includes residues Thr93, Ser95, Ser97, Phe147, Gln149,Ser151, Asn180, Thr182, Val251, Gln253, and Ser255. As used herein, thephrase “Cb atom” refers to the beta-carbon atom in the amino acidresidue side chain. The Cb atom is thus the first carbon in the proteinside chain that is present in all amino acid residues with the exceptionof Glycyl residues. With reference to FIG. 1, eHTP's can include, butare not limited to, one or more conservative or non-conservativesubstitutions of surface patch [1] amino acid residues T93, S95, S97,F147, Q149, S151, N180, T182, V251, Q253, and S255 or the equivalentamino acids within one or more of the scaffold proteins, particularlySEQ ID NO:2 (TIC807). eHTP's can include, but are not limited to, one ormore substitutions of surface patch [1] amino acid residues such as:T93A; S95A, S95V, S95L, or S95I; F147T, F147C, F147D, F147G, F147E,F147Y, F147M, F147N, F147Q, F147H, F147R, F147W, F147P, F147A, F147V,F147L, or F1471; Q149A, Q149C, Q149F, Q149E or Q149D; S151A; N180D;T182A; V251E or V251A, and/or Q253R. The other or second surface patchthat has been identified as amino acid residues that are receptive tomodifications which are result effective in conferring improvedHemipteran inhibitory bioactivity in the form of eHTP's of the presentinvention is defined as including the amino acid residues encompassedwithin a sphere having an atomic radius of from about 9.2 to about 12.2Angstroms (FIG. 2, sphere [2]) relative to the beta-carbon atom ofPro219 or the equivalent amino acid position in one or more of thescaffold proteins, particularly as set forth in SEQ ID NO:2, when anyone of the applicable scaffold proteins is folded into a threedimensional structure under physiological conditions, which includesresidues Val10, Ile14, Asn22, Asn23, Gly24, Ile25, Gln26, Gly27, Phe30,Gln38, Ile39, Asp40, Thr41, Ile43, Ser193, Thr194, Glu195, His196,Tyr197, Ser198, His199, Tyr200, Ser201, Gly202, Tyr203, Pro204, Ile205,Leu206, Thr207, Trp208, Ile209, Ser210, Tyr216, Ser217, Gly218, Pro219,Pro220, Met221, Ser222, Trp223, Tyr224, Phe225, Asn239, and Val244. SucheHTP's can include, but are not limited to, one or more conservative ornon-conservative amino acid residues substitutions and/or one or moreamino acid deletions within surface patch [2] including Val10, Ile14,Asn22, Asn23, Gly24, Ile25, Gln26, Gly27, Phe30, Gln38, Ile39, Asp40,Thr41, Ile43, Ser193, Thr194, Glu195, His196, Tyr197, Ser198, His199,Tyr200, Ser201, Gly202, Tyr203, Pro204, Ile205, Leu206, Thr207, Trp208,Ile209, Ser210, Tyr216, Ser217, Gly218, Pro219, Pro220, Met221, Ser222,Trp223, Tyr224, Phe225, Asn239, and Val244 of SEQ ID NO:2 (TIC807).eHTP's can include, but are not limited to, one or more substitutionsand/or deletions within the amino acid residues located within surfacepatch [2] such as: a deletion of any three contiguous amino acidresidues in the sequence His196, Tyr197, Ser198, His199, Tyr200, Ser201;Ser217Asn, Ser217Gln, Ser217Arg; and/or Pro219Arg, Pro219Asn, Pro219Gln.eHTP's can include, but are not limited to, one or more amino acidresidue substitutions and/or deletions within surface patch [2] such as:a deletion of any three contiguous HisTyrSer residues in the sequenceHis196, Tyr197, Ser198, His199, Tyr200, Ser201; Ser217Asn, Ser217Gln,Ser217Arg; and/or Pro219Arg, Pro219Asn, Pro219Gln. An eHTP can have atleast one amino acid modification in each of the two aforementionedsurface patches of the folded insect inhibitory protein. eHTP can haveone, or a combination of more than one modification at residues T93,S95, F147, Q149, S151, N180, T182, H196, Y197, S198, H199, Y200, S201,W208, S217, P219, W223, N239, V244, or V251 relative to SEQ ID NO:2(TIC807). Conservative amino acid changes can be made by substituting anacidic, basic, neutral polar, or neutral non-polar-type amino acid withanother amino acid of the same type. Non-conservative amino acid changescan be made by substituting an acidic, basic, neutral polar, or neutralnon-polar amino acid-type with an amino acid of a different type.Furthermore, of the eHTP proteins listed in Table 4B, all 267 are aminoacid sequence variants that exhibit increased toxicity to Lygus spp.when compared to one or more of the scaffold proteins, includingscaffold protein TIC807. Only ten of these amino acid sequence variantsexhibit modified amino acid residues compared to one or more of thescaffold proteins that are positioned outside of the two referencedsurface patches.

The prior art teaches solubility problems associated with the scaffoldproteins. eHTP's exhibit improved solubility compared to the scaffoldproteins, and generally exhibit increased solubility at a pH of lessthan 9.0, in contrast to the observed solubility profile of one or moreof the scaffold proteins. This increased solubility at morephysiological pH is evident when the eHTP is expressed in E. coli, in aplant cell, in a plant cell cytoplasm, a plant cell apoplast, or in ortargeted for import into a plastid of a plant cell. Amino acidmodifications that improve solubility relative to one or more of thescaffold proteins, including SEQ ID NO:2 (TIC807) include but are notlimited to, substitution of a lysine amino acid residue at one or moreof the following amino acid positions in TIC807 or the applicableresidue in any of the other scaffold proteins: 58, 59, 198, 199, 201, or202; or, substitution of a glutamic acid amino acid residue at one ormore of amino acid positions 198, 248 or 301; or, substitution of aarginine amino acid residue at one or more of amino acid positions 246,250 or 253.

Insect inhibitory compositions comprising the above described eHTP's arealso provided. Such compositions may further comprise at least oneadditional insect inhibitory agent different from the eHTP included inthe composition. The insect inhibitory agent is selected from any numberof insect inhibitory agents including an insect inhibitory protein, aninsect inhibitory dsRNA molecule, and one or more chemical agents usefulin controlling insect pests. Examples of additional inhibitory agentsincludes, but are not limited to, a TIC1415 protein, a dsRNA directedtowards Hemipteran orthologs of Nilaparvata lugens V-ATPase-E, 21E01, adsRNA directed towards Hemipteran orthologs of actin ortholog, ADP/ATPtranslocase, α-tubulin, ribosomal protein L9 (RPL9) or V-ATPase Asubunit, AXMI-171 (US20100298207A1), Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa,DIG11, DIG5, Cry7, eCry3.1Ab, mCry3A, Cry8, Cry34/Cry35, Cry3, DIG2,Cry1, Cry1A.105, Cry2, Cry1F, VIP3, 5307, and Cry9. Chemical agentsuseful in controlling Hemipteran species include but are not limited topyrethrins and synthetic pyrethroids; oxadizine derivatives;chloronicotinyls; nitroguanidine derivatives; triazoles;organophosphates; pyrrols; pyrazoles; phenyl pyrazoles;diacylhydrazines; biological/fermentation products; and carbamates.Known pesticides within these categories are listed in The PesticideManual, 11th Ed., C. D. S. Tomlin, Ed., British Crop Protection Council,Farnham, Surry, UK (1997).

Pyrethroids that are useful in the present composition includepyrethrins and synthetic pyrethroids. The pyrethrins that are preferredfor use in the present method include, without limitation,2-allyl-4-hydroxy-3-methyl-2-cyclopenten-1-one ester of2,2-dimethyl-3-(2methyl propenyl)-cyclopropane carboxylic acid, and/or(2-methyl-1-propenyl)-2-methoxy-4-oxo-3-(2 propenyl)-2-cyclopenten-1-ylester and mixtures of cis and trans isomers thereof (Chemical AbstractsService Registry Number (“CAS RN”) 8003-34-7).

Synthetic pyrethroids that are preferred for use in the presentinvention include (s)-cyano(3-phenoxyphenyl)methyl 4-chloro alpha(1-methylethyl)benzeneacetate (fenvalerate, CAS RN 51630-58-1),(S)-cyano (3-phenoxyphenyl) methyl (S)-4-chloro-alpha-(1-methylethyl)benzeneacetate (esfenvalerate, CAS RN 66230-04-4),(3-phenoxyphenyl)-methyl(+)cis-trans-3-(2,2-dichoroethenyl)-2,2-dimethylcyclopropanecarboxylate(permethrin, CAS RN 52645-53-1), (±) alpha-cyano-(3-phenoxyphenyl)methyl(+)-cis,trans-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate (cypermethrin, CAS RN 52315-07-8), (beta-cypermethrin, CASRN 65731-84-2), (theta cypermethrin, CAS RN 71697-59-1), S-cyano(3-phenoxyphenyl) methyl (±) cis/trans 3-(2,2-dichloroethenyl) 2,2dimethylcyclopropane carboxylate (zeta-cypermethrin, CAS RN 52315-07-8),(s)-alpha-cyano-3-phenoxybenzyl(IR,3R)-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropanecarboxylate(deltamethrin, CAS RN 52918-63-5), alpha-cyano-3-phenoxybenzyl2,2,3,3,-tetramethyl cyclopropoanecarboxylate (fenpropathrin, CAS RN64257-84-7),(RS)-alpha-cyano-3-phenoxybenzyl(R)-2-[2-chloro-4-(trifluoromethyl)anilino]-3-methylbutanoate(tau-fluvalinate, CAS RN 102851-06-9),(2,3,5,6-tetrafluoro-4-methylphenyl)-methyl-(1 alpha, 3alpha)-(Z)-(±)-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate(tefluthrin, CAS RN 79538-32-2), (±)-cyano (3-phenoxyphenyl) methyl(±)-4-(difluoromethoxy)-alpha-(1-methyl ethyl) benzeneacetate(flucythrinate, CAS RN 70124-77-5),cyano(4-fluoro-3-phenoxyphenyl)methyl3-[2-chloro-2-(4-chlorophenyl)ethenyl]-2,2-dimethylcyclopropanecarboxylate(flumethrin, CAS RN 69770-45-2), cyano(4-fluoro-3-phenoxyphenyl) methyl3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanedarboxylate(cyfluthrin, CAS RN 68359-37-5), (beta cyfluthrin, CAS RN 68359-37-5),(transfluthrin, CAS RN 118712-89-3),(S)-alpha-cyano-3-phenoxybenzyl(Z)-(IR-cis)-2,2-dimethyl-3-[2-(2,2,2-trifluoro-trifluoromethyl-ethoxycarbonyl)vinyl]cyclopropanecarboxylate (acrinathrin, CAS RN 101007-06-1), (IR cis) S and (IS cis) Renantiomer isomer pair ofalpha-cyano-3-phenoxybenzyl-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (alpha-cypermethrin, CAS RN 67375-30-8),[IR,3S)3(1′RS)(1′,2′,2′,2′-tetrabromoethyl)]-2,2-dimethylcyclopropanecarboxylic acid (s)-alpha-cyano-3-phenoxybenzyl ester(tralomethrin, CAS RN 66841-25-6), cyano-(3-phenoxyphenyl) methyl2,2-dichloro-1-(4-ethoxyphenyl)cyclopropane carboxylate (cycloprothrin,CAS RN 63935-38-6), [1α,3α(Z)]-(±)-cyano-(3-phenoxyphenyl)methyl3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-cimethylcyclopropanecarboxylate(cyhalothrin, CAS RN 68085-85-8), [1 alpha (s), 3alpha(z)]-cyano(3-phenoxyphenyl)methyl-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate (lambda cyhalothrin, CAS RN 91465-08-6), (2-methyl[1,1′-biphenyl]-3-yl) methyl3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl-cyclopropanecarboxylate(bifenthrin, CAS RN 82657-04-3),5-1-benzyl-3-furylmethyl-d-cis(1R,3S,E)2,2-dimethyl-3-(2-oxo,-2,2,4,5tetrahydro thiophenylidenemethyl)cyclopropane carboxylate (kadethrin,RU15525, CAS RN 58769-20-3), [5-(phenyl methyl)-3-furanyl]-3-furanyl2,2-dimethyl-3-(2-methyl-1-propenyl) cyclopropane carboxylate(resmethrin, CAS RN 10453-86-8),(1R-trans)-[5-(phenylmethyl)-3-furanyl]methyl2,2-dimethyl-3-(2-methyl-1-propenyl)cyclopropanecarboxylate(bioresmethrin, CAS RN 28434-01-7), 3,4,5,6-tetrahydro-phthalimidomethyl-(IRS)-cis-trans-chrysanthemate (tetramethrin,CAS RN 7696-12-0), 3-phenoxybenzyl-d,l-cis,trans2,2-dimethyl-3-(2-methylpropenyl) cyclopropane carboxylate (phenothrin,CAS RN 26002-80-2); (empenthrin, CAS RN 54406-48-3); (cyphenothrin; CASRN 39515-40-7), (prallethrin, CAS RN 23031-36-9), (imiprothrin, CAS RN72963-72-5), (RS)-3-allyl-2-methyl-4-oxcyclopent-2-enyl-(1A,3R;1R,3S)-2,2-dimethyl-3-(2-methylprop-1-enyl) cyclopropane carboxylate(allethrin, CAS RN 584-79-2), (bioallethrin, CAS RN 584-79-2), and(ZXI8901, CAS RN 160791-64-0). It is believed that mixtures of one ormore of the aforementioned synthetic pyrethroids can also be used in thepresent invention. Particularly preferred synthetic pyrethroids aretefluthrin, lambda cyhalothrin, bifenthrin, permethrin and cyfluthrin.Even more preferred synthetic pyrethroids are tefluthrin and lambdacyhalothrin, and yet more preferred is tefluthrin.

Insecticides that are oxadiazine derivatives are useful in the subjectinvention. The oxadizine derivatives that are preferred for use in thepresent invention are those that are identified in U.S. Pat. No.5,852,012. More preferred oxadiazine derivatives are5-(2-chloropyrid-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazine,5-(2-chlorothiazo-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazine,3-methyl-4-nitroimino-5-(1-oxido-3-pyridinomethyl)perhydro-1,3,5-oxadiazine,5-(2-chloro-1-oxido-5-pyridiniomethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxidiazine;and3-methyl-5-(2-methylpyrid-5-ylmethyl)-4-nitroiminoperhydro-1,3,5-oxadiazine.Even more preferred is thiamethoxam (CAS RN 153719-23-4).

Chloronicotinyl insecticides are also useful in the subject invention.Chloronicotinyls that are preferred for use in the subject compositionare described in U.S. Pat. No. 5,952,358, and include acetamiprid((E)-N-[(6-chloro-3-pyridinyl)methyl]-N′-cyano-N-methyleneimidamide, CASRN 135410-20-7), imidacloprid(1-[(6-chloro-3-pyridinyl)methol]-N-nitro-2-imidazolidinimime, CAS RN138261-41-3), and nitenpyram(N-[(6-chloro-3-pyridinyl)methyl]-N-ethyl-N′-methyl-2-nitro-1,1-ethenediamine,CAS RN 120738-89-8).

Nitroguanidine insecticides are useful in the present invention. Suchnitroguanidines can include those described in U.S. Pat. Nos. 5,633,375,5,034,404 and 5,245,040.

Pyrrols, pyrazoles and phenyl pyrazoles that are useful in the presentinvention include those that are described in U.S. Pat. No. 5,952,358.Preferred pyrazoles include chlorfenapyr(4-bromo-2-(4-chlorophenyl)-1-ethoxymethyl-5-trifluoromethylpyrrole-3-carbonitrile,CAS RN 122453-73-0), fenpyroximate((E)-1,1-dimethylethyl-4[[[[(1,3-dimethyl-5-phenoxy-1H-pyrazole-4-yl)methylene]amino]oxy]methyl]benzoate,CAS RN 111812-58-9), and tebufenpyrad(4-chloro-N[[4-1,1-dimethylethyl)phenyl]methyl]-3-ethyl-1-methyl-1H-pyrazole-5-carboxamide,CAS RN 119168-77-3). A preferred phenyl pyrazole is fipronil(5-amino-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(1R,S)-(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile,CAS RN 120068-37-3).

Diacylhydrazines that are useful in the present invention includehalofenozide(4-chlorobenzoate-2-benzoyl-2-(1,1-dimethylethyl)-hydrazide, CAS RN112226-61-6), methoxyfenozide (RH-2485;N-tert-butyl-N′-(3-methoxy-o-toluoyl)-3,5-xylohydrazide, CAS RN161050-58-4), and tebufenozide (3,5-dimethylbenzoic acid1-(1,1-dimethylethyl)-2,(4-ethylbenzoyphydrazide, CAS RN 112410-23-8).

Triazoles, such as amitrole (CAS RN 61-82-5) and triazamate are usefulin the method of the present invention. A preferred triazole istriazamate (ethyl[[1-[(dimethylamino)carbonyl]-3-(1,1-dimethylethyl)-1H-1,2,4-triazol-5-yl]thio]acetate,CAS RN 112143-82-5).

Biological/fermentation products, such as avermectin (abamectin, CAS RN71751-41-2) and spinosad (XDE-105, CAS RN 131929-60-7) are useful in thepresent invention.

Organophosphate insecticides are also useful as one of the components ofthe present invention. Preferred organophophate insecticides includeacephate (CAS RN 30560-19-1), chlorpyrifos (CAS RN 2921-88-2),chlorpyrifos-methyl (CAS RN 5598-13-0), diazinon (CAS RN 333-41-5),fenamiphos (CAS RN 22224-92-6), and malathion (CAS RN 121-75-5).

In addition, carbamate insecticides are useful in the subject invention.Preferred carbamate insecticides are aldicarb (CAS RN 116-06-3),carbaryl (CAS RN 63-25-2), carbofuran (CAS RN 1563-66-2), oxamyl (CAS RN23135-22-0) and thiodicarb (CAS RN 59669-26-0).

When a chemical insecticide is described herein, it is to be understoodthat the description is intended to include salt forms of theinsecticide as well as any isomeric and/or tautomeric form of theinsecticide that exhibits the same insecticidal activity as the form ofthe insecticide that is described.

The chemical insecticides that are useful in the present invention canbe of any grade or purity that pass in the trade as such insecticide.Other materials that accompany the insecticides in commercialpreparations as impurities can be tolerated in the subject invention andcompositions, as long as such other materials do not destabilize thecomposition or significantly reduce or destroy the activity of any ofthe insecticide components or the transgenic event against the targetpest(s). One of ordinary skill in the art of the production ofinsecticides can readily identify those impurities that can be toleratedand those that cannot.

eHTP's are related by amino acid modifications such that the modifiedproteins exhibit enhanced Hemipteran inhibitory spectrum and/or improvedHemipteran inhibitory activity against Lygus spp., Empoasca spp. and/orAmrasca spp. compared to the parent protein, TIC807. The phrases “moreactive”, “improved activity”, “enhanced specificity”, “increased toxicpotency”, “increased toxicity”, “improved Hemipteran inhibitoryactivity, “enhanced Hemipteran inhibitory activity”, “improved Lygus,Empoasca and/or Amrasca inhibitory activity”, “greater Lygus, Empoascaand/or Amrasca inhibitory activity”, “greater Hemipteran inhibitoryactivity” and “enhanced Lygus, Empoasca and/or Amrasca inhibitoryspectrum” and “enhanced Hemipteran inhibitory spectrum” refer to acomparison of the activity of an eHTP and of the activity of a TIC807(SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ ID NO:182), TIC853(SEQ ID NO:184), and/or a AXMI-171 (SEQ ID NO:206) protein against aHemipteran insect, wherein activity attributed by the eHTP of thepresent invention is greater than the activity attributed to the TIC807protein (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ IDNO:182), TIC853 (SEQ ID NO:184, and/or a AXMI-171 (SEQ ID NO:206)protein. eHTP's provided herein exhibit enhanced Hemipteran inhibitoryspectrum and/or improved or greater Hemipteran inhibitory activity whencompared to the Bacillus thuringiensis proteins of SEQ ID NO:2, SEQ IDNO:8, SEQ ID NO:182, and SEQ ID NO:184, where the Hemipteran pestspecies include Lygus hesperus, Lygus lineolaris, Empoasca fabae, andAmrasca devastans. Amrasca devastans is also called Amrasca biguttulabiguttula. eHTP's exhibiting enhanced insect inhibitory spectrum and/orimproved insect inhibitory activity compared to TIC807 can be identifiedby many different methods. In general, exemplary and non-limitingmethods for identifying eHTP proteins can comprise:

-   -   (1) administering identical amounts of a test eHTP and of        control TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1        (SEQ ID NO:182), TIC853 (SEQ ID NO:184), and/or an AXMI-171 (SEQ        ID NO:206) protein to a test insect under controlled assay        conditions; and, measuring and comparing the potency of the test        and control proteins; and/or,    -   (2) determining the protein doses (e.g., protein concentration        in diet) of a test eHTP and of control TIC807 (SEQ-ID NO:2),        TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ ID NO:182), TIC853 (SEQ        ID NO:184), and/or an AXMI-171 (SEQ ID NO:206) protein which        elicit equivalent insect population responses under controlled        assay conditions (i.e. obtaining a dose response curve).        In the second approach, a statistically robust dose response        value used for comparison would be the median lethal        concentration (LC50) required to kill 50% of a test population.        However, in certain embodiments, other values including but not        limited to, a median inhibitory concentration (“IC50”) required        to result in 50% growth inhibition of a test population can be        used. In this context, “growth inhibition” can comprise stunting        and/or inhibition of Hemipteran development.

As used herein, the phrase “an insect inhibitory amount”, refers to anamount of a composition containing an agent that is effective inachieving any measurable inhibition of insect viability, growth, insectdevelopment, insect reproduction, insect feeding behavior, insect matingbehavior and/or any measurable decrease in the adverse effects caused byinsect feeding on a composition containing the agent. Similarly, a“Hemipteran inhibitory amount” refers to an amount of a protein of thepresent invention alone or with other agents targeting the applicableHemipteran species for control, that results in any measurableinhibition of target insects belonging to the order Hemiptera related toviability, growth, development, reproduction, feeding behavior, matingbehavior, and or any measurable decrease in the adverse effects causedby Hemipteran insects feeding on a plant. Likewise, “Lygus, Empoascaand/or Amrasca inhibitory amount” refers to an amount of a compositioncontaining one or more proteins of the present invention, i.e., eHTP's,or other agent that results in any measurable inhibition, viability,growth, development, reproduction, feeding behavior, mating behaviorand/or any measurable decrease in the adverse effects caused by Lygus,Empoasca and/or Amrasca feeding on a composition containing that eHTP.As used herein in the context of an eHTP, an “enhanced Hemipteraninhibitory activity or “greater enhanced Hemipteran inhibitory activity”refers to any measurable increase in the inhibition of Hemipteranviability, growth, development, reproduction, feeding behavior, matingbehavior and/or any measurable decrease in the adverse effects caused byHemipteran feeding on a composition containing that eHTP relative to thecorresponding inhibitory activity observed with any one or more of thescaffold proteins, including TIC807, Cry51Aa1 (SEQ ID NO:182), TIC853(SEQ ID NO:184), and/or AXMI-171 (SEQ ID NO:206) proteins. Likewise,“enhanced Lygus, Empoasca and/or Amrasca inhibitory activity” or“greater enhanced Lygus, Empoasca and/or Amrasca inhibitory activity”refers to any measurable increase in the inhibition, viability, growth,development, reproduction, feeding behavior, mating behavior and/or anymeasurable decrease in the adverse effects caused by the presence of oneor more eHTP of the present invention in a composition or plant providedin the diet of Lygus, Empoasca and/or Amrasca relative to thecorresponding inhibitory activity observed with an equivalentcomposition or plant containing only an applicable amount of one or moreof the scaffold proteins, including but not limited to TIC807 (SEQ IDNO:2), Cry51Aa1 (SEQ ID NO:182), TIC853 (SEQ ID NO:184), and/or AXMI-171(SEQ ID NO:206) proteins.

As used herein in the context of an eHTP, an “enhanced Lygus, Empoascaand/or Amrasca inhibitory spectrum” refers to any measurable increase inthe inhibition of a specific Lygus spp., Empoasca spp. and/or Amrascaspp. viability, growth, development, reproduction, feeding behavior,mating behavior and/or any measurable decrease in the adverse effectscaused by that Lygus spp., Empoasca spp. and/or Amrasca spp. feeding ona plant relative to the corresponding inhibition of that specific Lygusspp., Empoasca spp. and/or Amrasca spp. observed with the TIC807protein. In certain embodiments, eHTP provided herein exhibit anenhanced Lygus inhibitory spectrum relative to TIC807 in that thoseeHTP's can provide increased inhibition of Lygus lineolaris.

An eHTP provided herein can exhibit from about 2 to about 260 foldgreater Lygus, Empoasca and/or Amrasca inhibitory activity against aLygus, Empoasca and/or Amrasca pest species than a protein of SEQ IDNO:2 (TIC807), SEQ ID NO:8 (TIC807_M2), SEQ ID NO:182 (Cry51Aa1), SEQ IDNO:184 (TIC853), and SEQ ID NO:206 (AXMI-171). An eHTP provided hereincan exhibit from about 3, 4, 5, 7, 8, 10, 12, 15, 20, 25, 27, 30, 38,46, 50, 52, 54, 66, 91, 122, 186, 243, or 262 fold greater Lygus,Empoasca and/or Amrasca inhibitory activity against a Lygus, Empoascaand/or Amrasca pest species than a protein of SEQ ID NO:2 (TIC807), SEQID NO:8 (TIC807_M2), SEQ ID NO:182 (Cry51Aa1), SEQ ID NO:184 (TIC853),and SEQ ID NO:206 (AXMI-171).

eHTP's can exhibit an enhanced target pest inhibitory spectrum and/orimproved target pest inhibitory activity over SEQ ID NO:2 (TIC807), SEQID NO:8 (TIC807_M2), SEQ ID NO:182 (Cry51Aa1), SEQ ID NO:184 (TIC853),and/or a SEQ ID NO:206 (AXMI-171) by causing mortality:

-   -   (i) at a dose of about 0.3 μg/mL to about 70 μg/mL against a        Lygus hesperus insect species,    -   (ii) at a dose of about 0.85 μg/mL to about 100 μg/mL against a        Lygus lineolaris insect species,    -   (iii) measuring at an LC50 value of about 0.3 to about 70 μg/mL        against Lygus hesperus,    -   (iv) measuring at an LC50 value of about 0.85 to about 100 μg/mL        against Lygus lineolaris, or    -   (v) measuring at an LC50 value of more than two-fold lower the        LC50 value of TIC807, SEQ ID NO:8, SEQ ID NO:182 (Cry51Aa1), SEQ        ID NO:184 (TIC853), and/or a SEQ ID NO:206 (AXMI-171) against        Lygus spp, Emrasca spp. and/or Amrasca spp., or    -   (vi) at a dose of about 0.69 μg/mL to about 500 μ/mL against a        Amrasca devastans or Empoasca fabae insect species, or    -   (vii) measuring at an LC50 value of about 3.5 to about 15 μg/mL        against Amrasca devastans and/or Empoasca fabae.

Table 4A and 4B tabulate the exemplary eHTP's of the present inventionwith Amrasca and Lygus spp. mortality data. Mortality data available forLygus spp. and Amrasca spp. are reported either as (a) a μg/mL LC50value, or as (b) a % mortality at doses of about 1 to about 3 μg/mL forL. hesperus or about 100 μg/mL protein for L. lineolaris, and about 0.69to 500 μg/mL for Amrasca devastans. The fold increased toxicity comparedto TIC807 (SEQ ID NO:2) and TIC807_M2 (SEQ ID NO:8) is provided forexemplary eHTP's where LC50 values were determined.

The eHTP's of the present invention are particularly useful incontrolling insects of the order Hemiptera compared to the scaffoldproteins. Lygus lineolaris required high doses of TIC807 protein (e.g.,in excess of 100 μg/mL) to elicit mortality. The dose response curve forone eHTP of the present invention TIC807_M8 (SEQ ID NO:16), an eHTP thatexhibits remarkably improved toxic effects against both L. lineolarisand L. hesperus, but against L. lineolaris the eHTP exhibits acalculated LC50 value of 223 μg/mL. It has not been possible previouslyto achieve a protein concentration toxic dose that can elicit greaterthan 50% mortality against L. lineolaris species because providingsignificantly large doses of TIC807 and TIC807_M2 protein in excess of1000 μg/mL in the diet has not been possible. Therefore, LC50 valuesagainst L. lineolaris for TIC807 and TIC807_M2 (SEQ ID NO:8) proteinswere not determined, but rather estimated as greater than (>) 223 μg/mL(See Tables 1 and 3, Example 4, and FIG. 1B).

Iterative design refers to a semi-random approach for developing andselecting eHTP's including a combination of engineering, testing, andselecting (not necessarily in that order) (see Examples 1 through 4).The word “engineering” is intended to include identifying relevantresidues to modify, cloning, and expressing eHTP's described herein. Theword “testing” is intended to refer to comparing the Hemipteran activityof an eHTP to the activity of a scaffold protein such as TIC807 (SEQ IDNO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ ID NO:182), and/or TIC853(SEQ ID NO:184); or, comparing an eHTP of the present invention againstanother protein such as AXMI-171 (SEQ ID NO:206). The word “selecting”is intended to refer to the act of identifying improved variant proteinsof the present invention, i.e., eHTP's, and the applicable amino acidresidues for “engineering”.

Iterative design includes the elucidation of the atomic structure ofproteins of the present invention (for example, as set forth in FIG. 2)and the use of the atomic structure to guide and complement semi-randomapproaches of “selecting” amino acid residues to modify for“engineering”, and in this case, has included the identification ofamino acid residues at loops and at surface exposed regions of a foldedinsect inhibitory scaffold protein such as TIC807, TIC853, and Cry51Aa1that can be modified to confer improvements to insect inhibitoryspectrum and activity. Such amino acid residues at loops and at surfaceexposed regions are selected for “engineering”. In this case, iterativedesign has included the identification of two different regions withinthe three dimensional structure of the scaffold protein that harbor anaccumulation of relevant amino acid residues that, when modified tocontain amino acid residues other than those appearing at thosepositions in the naturally occurring scaffold protein, result in one ormore of the eHTP proteins of the present invention.

Initially the scaffold protein TIC807 (SEQ ID NO:2) used in this processof iterative design, and 267 different eHTP's were discovered thatexhibited increased Lygus spp. inhibitory activity compared to thescaffold protein TIC807. TIC807_M8 (SEQ ID NO:16) was discovered inearly rounds of the design process. Subsequent rounds of iterativeengineering-testing-selecting led to the discovery of other eHTPproteins that exhibited yet greater levels of toxicity against Lygusspecies and also exhibited a broader host range of toxic effects whencompared to the scaffold protein. Seven variants (eHTP's) exhibitedsignificantly higher levels of increased toxicity against both Lygusspecies (L. hesperus and L. lineolaris) when compared to TIC807. LC50values for these seven, and other, eHTP's constructed herein weredetermined against Lygus hesperus and Lygus lineolaris species andcompared to LC50 values for scaffold proteins, particularly TIC807. Theresults are shown in Table 1, and FIG. 3 is a bar chart showinggraphically the results observed as tabulated in Table 1.

TABLE 1 LC50 values of select eHTP's compared to TIC807 Lygus hesperusLygus lineolaris SEQ LC50 Toxicity LC50 Toxicity ID value (fold value(fold NO: Toxin (μg/mL) increase) (μg/mL) increase) 2 TIC807 73 1 >223*1 6 TIC807_M1 23 3 100 ≥2 8 TIC807_M2 5.9 12 >223* ~1 10 TIC807_M3 2.925 ND — 12 TIC807_M4 2.4 30 ND — 14 TIC807_M5 1.1 66 ND — 18 TIC807_M61.45 50 ND — 20 TIC807_M7 1.4 52 ND — 16 TIC807_M8 0.8 91 223 ≥1 28TIC807_M9 9.9 7    8.3 ≥27 30 TIC807_M10 0.6 122    4.8 ≥46 32TIC807_M11 1.35 54    5.9 ≥38 36 TIC807_M12 0.4 182    1.2 ≥186 34TIC807_M13 0.3 243    0.85 ≥262 ND = Not Determined. LC50 values aredetermined by presenting 8-10 different protein concentrations to apopulation of newly hatched Lygus nymphs, allowing nymphs to feed for 5days, and then scoring for mortality over the dose range provided.*Toxicity, displayed in terms of a multiple of increased activitycompared to the level observed against Lygus hesperus using the observedLD50 for TIC807 as the baseline value of 1. Significantly large amountsof protein in excess of 1000 μg/mL have not been possible to provide inLygus diet in order to complete the high range of toxicity dose responseto Lygus lineolaris. Therefore, an LC50 value was not determined forTIC807 or TIC807_M2. Instead, a 4-dose LC50 estimation in the low rangewas performed verifying that expected LC50 values for TIC807 andTIC807_M2 are greater than 223 μg/mL.

With reference to Table 1, the iterative design process has provided ameans for identifying proteins exhibiting improved toxic properties, notonly to Lygus hesperus, but also to Lygus lineolaris.

Recombinant polynucleotide compositions that encode eHTP's are alsoprovided. In certain embodiments, eHTP's can be expressed withrecombinant DNA constructs in which a polynucleotide molecule with theopen reading frame encoding the protein is operably linked to elementssuch as a promoter and any other regulatory element functional forexpression in the system for which the construct is intended. Forexample, plant-functional promoters can be operably linked to anapplicable eHTP coding sequence to enable expression of the protein inplants. Promoters functional in bacteria are also contemplated for usein expression cassettes. Promoters functional in an applicablebacterium, for example, in an E. coli or in a Bacillus thuringiensisspecies can be operably linked to the eHTP coding sequences forexpression of the applicable protein in the applicable bacterial strain.Other useful elements that can be operably linked to the eHTP codingsequences include, but are not limited to, enhancers, introns, leaders,encoded protein immobilization tags (HIS-tag), encoded sub-cellulartranslocation peptides (i.e. plastid transit peptides, signal peptides),encoded polypeptide sites for post-translational modifying enzymes,ribosomal binding sites, and segments designed for use as RNAi triggersfor suppression of one or more genes either in plants or in a particulartarget pest species.

Exemplary recombinant polynucleotide molecules provided herein include,but are not limited to, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193,SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ IDNO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:35, and SEQ ID NO:201.These sequences encode the respective proteins each having the aminoacid sequence as set forth in SEQ ID NO:4 (TIC807_4), SEQ ID NO:6(TIC807_M1), SEQ ID NO:8 (TIC807_M2), SEQ ID NO:10 (TIC807_M3), SEQ IDNO:12 (TIC807_M4), SEQ ID NO:14 (TIC807_M5), SEQ ID NO:16 (TIC807_M8),SEQ ID NO:18 (TIC807_M6), SEQ ID NO:20 (TIC807_M7), SEQ ID NO:22(TIC807_22), SEQ ID NO:24 (TIC807_24), SEQ ID NO:26 (TIC807_26), SEQ IDNO:28 (TIC807_M9), SEQ ID NO:30 (TIC807_M10), SEQ ID NO:32 (TIC807_M11),SEQ ID NO:36 (TIC807_M12), and SEQ ID NO:34 (TIC807_M13). Because of theredundancy of the genetic code, the codons of a recombinantpolynucleotide molecule encoding for proteins of the present inventionmay be substituted for synonymous codons (also called a silentsubstitution); and are within the scope of the present invention.Recombinant polynucleotides encoding any of the eHTP's disclosed hereinare thus provided.

A recombinant DNA construct comprising eHTP coding sequences can alsofurther comprise a region of DNA that codes for one or more insectinhibitory agents which can be configured to be co-expressed along witha DNA sequence encoding an applicable eHTP, a protein different from aneHTP, or an insect or plant gene inhibitory dsRNA molecule. Arecombinant DNA construct can be assembled so that all agents designedto be expressed from a particular construct are expressed from onepromoter or so that separate agents are each under separate promotercontrol, or some combination thereof. The proteins of this invention canbe expressed from a multi-gene expression system in which one or moreproteins are expressed from a common nucleotide segment on which is alsocontained other open reading frames and/or promoters depending on thetype of expression system selected.

Recombinant polynucleotide or recombinant DNA construct comprising aneHTP encoding sequence can be delivered to host cells by vectors, e.g.,a plasmid, baculovirus, artificial chromosome, virion, cosmid, phagemid,phage, or viral vector. Such vectors can be used to achieve stable ortransient expression of an eHTP encoding sequence in a host cell; and,if the case may be, subsequent expression to polypeptide. An exogenousrecombinant polynucleotide or recombinant DNA construct that comprisesan eHTP encoding sequence and that is introduced into a host cell isalso referred to herein as a “transgene”.

Also provided herewith are transgenic bacteria, transgenic plant cells,transgenic plants, and transgenic plant parts that contain any arecombinant polynucleotide (i.e. transgene) that expresses any one ormore eHTP encoding sequence. It is intended that “bacterial cell” or“bacterium” can include, but are not limited to, an Agrobacterium, aBacillus, an Escherichia, a Salmonella, a Pseudomonas, or a Rhizobiumcell. It is intended that “plant cell” or “plant” include an alfalfa,almont, banana, barley, bean, beet, broccoli, cabbage, brassica,brinjal, carrot, cassava, castor, cauliflower, celery, chickpea, Chinesecabbage, celery, citrus, coconut, coffee, corn, clover, cotton, acucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic,grape, guar, hops, leek, legumes, lettuce, Loblolly pine, millets,melons, nectarine, nut, oat, okra, olive, onion, ornamental, palm,pasture grass, papaya, pea, peach, peanut, pepper, pigeonpea, pine,potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice,rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweetcorn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato,triticale, turf grass, watermelon, and wheat plant cell or plant. Incertain embodiments: transgenic plants and transgenic plant partsregenerated from a transgenic plant cell are provided; transgenic plantscan be obtained from a transgenic seed; transgenic plant parts can beobtained by cutting, snapping, grinding or otherwise disassociating thepart from the plant; the plant part can be a seed, a boll, a leaf, aflower, a stem, a root, or any portion thereof; and a transgenic plantpart provided herein is a non-regenerable portion of a transgenic plantpart. As used in this context, a “non-regenerable” portion of atransgenic plant part is a portion that can not be induced to form awhole plant or that can not be induced to form a whole plant that iscapable of sexual and/or asexual reproduction. A non-regenerable portionof a plant part is a portion of a transgenic pollen, ovule, seed, boll,leaf, flower, stem, or root.

Also provided herein are methods of making transgenic plants thatcontain insect or Lygus and/or Amrasca inhibitory amounts of an eHTP.Such plants can be made by introducing a recombinant polynucleotide thatencodes any of the eHTP proteins provided herein into a plant cell, andselecting a plant derived from said plant cell that expresses an insector Hemipteran inhibitory amount of the eHTP's. Plants can be derivedfrom the plant cells by regeneration, seed, pollen, or meristemtransformation techniques.

Transgenic plants and host cells are provided that expresse an insect orHemipteran inhibitory amount of the eHTP to control an insect orHemipteran infestation. Any of the aforementioned plant species can beused for protecting a plant from insect or Hemipteran infestationprovided herein as long as the plant is transformed with apolynucleotide construct designed to express the applicable eHTP.

Additional aspects of the invention include antibodies, kits, methodsfor detecting polynucleotides that encode eHTP's or distinguishingfragments thereof, or eHTP's or distinguishing fragments thereof,methods for identifying additional insect inhibitory members of theprotein genus of the present invention, formulations and methods forcontrolling insect growth and/or infestation, and methods for providingsuch control to plants and other recipient hosts. Each composition,construct, cell, plant, formulation, method or kit provides for theindustrial application of the proteins of the present invention, forexample, by increasing plant productivity through the commercial use ofany of these proteins to inhibit insects.

A plant product, other than a seed or a fruit or vegetable, is intendedas a commodity or other products which move through commerce and arederived from a transgenic plant or transgenic plant part, in which thecommodity or other products can be tracked through commerce by detectingnucleotide segments, RNA or proteins that corresponding to an eHTP ofthe present invention and are produced in or maintained in the plant orplant tissue or part from which the commodity or other product has beenobtained. Such commodity or other products of commerce include, but arenot limited to, plant parts, biomass, oil, meal, sugar, animal feed,flour, flakes, bran, lint, processed seed, and seed. Plant parts includebut are not limited to a plant seed, boll, leaf, flower, stem, pollen,or root. In certain embodiments, the plant part is a non-regenerableportion of said seed, boll, leaf, flower, stem, pollen, or root. Cottonand flax plant bolls and non-regenerable portions thereof that containthe eHTP's are also provided.

Also provided herewith are processed plant products that contain adetectable amount of an eHTP, an insect inhibitory fragment thereof, orany distinguishing portion thereof. Without seeking to be limited bytheory, it is believed that such processed plant products containing adetectable amount of one or more of the eHTP's provided herein can incertain embodiments exhibit reductions in undesirable microorganismsthat can be transmitted by Hemiptera and/or reductions in theundesirable side products of such microorganisms. In certainembodiments, a distinguishing portion thereof can comprise anypolypeptide of at least from about 20 to about 100 or more contiguousamino acids as set forth in SEQ ID NO:180, in particular in which thepolypeptide does not contain a corresponding polypeptide of contiguousamino acids present in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:182, or SEQID NO:184, and wherein the polypeptide comprises at least one amino acidsubstitution, addition, or deletion in the corresponding amino acidsequence as set forth in SEQ ID NO:2.

Processed plant products are provided that contain a detectable amountof a recombinant polynucleotide encoding an eHTP, an eHTP or an insectinhibitory fragment thereof, or any distinguishing portion thereof. Theprocessed product is selected from the group consisting of plantbiomass, oil, meal, animal feed, flour, flakes, bran, lint, hulls, andprocessed seed.

Hemiptera infestations of crop plants are controlled by providing in thecrop plants a recombinant polynucleotide sequence encoding one or moreof the eHTP's of the present invention. Such transgenic crops produce orare treated to contain an insect or Hemiptera inhibitory amounst of anapplicable eHTP, and such crops are imbued with sufficient eHTP by (i)applying any composition comprising or encoding an eHTP to the plant ora seed that gives rise to the plant; and/or (ii) transforming the plantor a plant cell that gives rise to the seed and ultimately, the plant,with a polynucleotide encoding an eHTP. The plant may be a transientlyor stably transformed transgenic plant comprising a transgene thatexpresses an insect or Hemiptera inhibitory amount of an eHTP. The plantmay be a non-transgenic plant to which a composition comprising an eHTPhas been applied. In such methods, the plant is a dicot plant, and morespecifically may be a cotton, soybean or alfalfa plant. The Hemipteraninsects include adults and nymphs.

Preferably, the Lygus spp. is Lygus hesperus or Lygus lineolaris, theEmpoasca spp. is Empoasca fabae, and the Amrasca spp. is Amrascadevastans.

Other features and advantages of the invention will be apparent from thefollowing detailed description, examples, and claims.

EXAMPLES

In view of the foregoing, those of skill in the art should appreciatethat changes can be made in the specific aspects which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention. Thus, specific details disclosed herein arenot to be interpreted as limiting. The U.S. Provisional Application Ser.No. 61/621,436 to which this application claims the benefit of priority,the Sequence Listing, as well as all references material to theinventions disclosed and claimed, particularly references and publishedpatent applications cited in this application, are incorporated hereinby reference in their entirety.

Example 1: Iterative Engineering-Testing-Selecting Approach

This example illustrates the random, combinatorial, and inventiveaspects of the iterative (also can be referred to as “recursive”)engineering-testing-selecting approach used to identify and describeinsect inhibitory proteins exhibiting Coleopteran and/or nematicidalactivity or increased toxicity to Hemipteran insect species compared toTIC807 (SEQ ID NO:2). Several design approaches were employed toengineer for eHTP's with greater inhibitory activity against Lygusspecies; approaches that included but were not limited to semi-randommodifications, directed modifications of variances in an alignment ofTIC807 with other native Bt proteins, and structure/function assisteddesign. Numerous rounds of engineering and testing were conducted (bothconsecutively and concurrently) to select for TIC807 protein variantsexhibiting increased toxicity. Design approaches were adjusted as datawas collected. This iterative engineering-testing-selection approachalso included, but was not limited to steps including cloning,expressing, purifying, and bioassay testing of TIC807 control proteincompared to the eHTP's.

About 267 exemplary eHTP's having exhibited increased Lygus toxicitycompared to TIC807 were obtained from more than 2000 groups of candidateeHTP's (i.e. “test” proteins) that were assayed for improved insectinhibitory activity. The actual total number of candidate eHTP's testedwas much greater than 2000 because testing included recombinantnucleotide segments encoding a number of candidate eHTP's derived fromlibrary mutagenesis that were not sequenced in the selection process.

Protein stocks of various amounts and purity were prepared depending onthe purpose of the test and the testing throughput desired. For example,lower quantity and lower purity protein preparations were prepared forscreening higher numbers of variants in bioassay. Larger quantity andhigher purity protein stocks were prepared for high-powered bioassays.Testing trended towards the high-powered bioassays as principallyrelevant residue positions of the improved variants were elucidated.Initially, about 2000 variants were tested on Lygus hesperus. Based ondata from L. hesperus approximately 600 variants were designed and thenfurther tested on Lygus lineolaris. Of these, about 267 variants (Table4B) demonstrated increased toxicity against Lygus hesperus and/or Lyguslineolaris when compared to TIC807. These 267 variants includedtwenty-two (22) variants that were confirmed to demonstrate increasedtoxicity against both Lygus species. Further confirmation and doseresponse testing narrowed the selection to seven (7) variants that weresubsequently characterized using an 8-dose replicated bioassay todetermine LC50 values against both Lygus species.

The selection process included dynamic updates of testing data,constantly adjusting engineering approaches, and performing iterativerounds. Concurrently, labor intensive cloning, protein expression,protein purification, and bioassay experiments were employed test thecandidate eHTP's.

Example 2: Engineering Approaches

Alignment Based Approaches

A multiple sequence alignment of protein members of Cry51: Cry51Aa1 (SEQID NO:182), TIC853 (SEQ ID NO:184), and TIC807 (SEQ ID NO:2) were usedto identify regions of variability, e.g., positions 195 to 201 andpositions 211 to 219, relative to SEQ ID NO:2 (TIC807). These regionswere targeted for saturation mutagenesis through use of degenerateoligonucleotide primers encoding random amino acid residues in theseregions. Construct libraries were prepared for subsequent proteinexpression in host cells.

A multiple sequence alignment of Cry51Aa1 (SEQ ID NO:182), TIC853 (SEQID NO:184), and TIC807 (SEQ ID NO:2) was used in combination with aBLOSUM 80 substitution matrix to calculate average pair-wise distancesfor each position variant to TIC807. Residue positions with loweraverage pair-wise distances were substituted with alternative amino acidresidues using degenerate oligonucleotide primers encoding foralternative amino acid residues, e.g., G28X, G31X, F46X, E125X, F138X,F147X, S167X, Y216X, P218X, G234X, T247X, D268X, and T308X. Constructlibraries were prepared for subsequent protein expression in host cells.

Scanning Approaches

Polynucleotide constructs were engineered to express a single Alaninesubstitution or a double Alanine substitution (Alanine-<parentresidue>-Alanine) at every possible position over the full-length of SEQID NO:2 (TIC807). See Table 2 for a hypothetical example.

TABLE 2 A hypothetical example of single and double Alanine scans on ascaffold protein containing the amino acid sequence XXXXAXX. SingleAlanine Scan Double Alanine Scan 1 AXXXaXX AXAXaXX 2 XAXXaXX XAXAaXX 3XXAXaXX XXAXSXX 4 XXXAaXX XXXAaAX 5 XXXXSXX XXXXSXA 6 XXXXaAX — 7XXXXaXA — X = parent residue a = parent residue is an Alanine residue A= Modified to an Alanine residue S = Modified to a Serine residue

Where an Alanine residue was already present in TIC807, a Serine wassubstituted instead. Protein variants that exhibited increased toxicitycompared to TIC807 were further tested by combination and saturationmutagenesis at those Alanine-substituted residues that conferredincreased toxicity. Scanning approaches were also performed on improvedcombination variants having accumulated modifications from previousiterative rounds of engineering-testing-selecting, e. g., TIC807_M2 (SEQID NO:8) having mutations F46S, Y54H, S167R, S217N, and a contiguoustriple deletion in residue range 196-201 was further engineered by anadditional round of single Alanine substitutions to further improve uponthe improved TIC807_M2. Principally relevant residues were identifiedand further tested by combination and saturation mutagenesis (e. g.,A150X, E125X, E155X, F147X, I134X, N157X, Q149X, T133X, E135X, andN137X). Variants engineered by these combined approaches exhibitedfurther improvements to increased toxicity compared to TIC807 and werefurther combined with other design approaches that took advantage of theatomic structure of TIC807 (SEQ ID NO:2).

Surface Exposed Residues

The atomic structure of proteins of the present invention was determinedin the midst of the Iterative Engineering-Testing-Selecting approach;and, the relative solvent-accessibility (% SA) of each residue wasdetermined using Molsoft's ICM-Browser (Molsoft L.L.C., 11199 SorrentoValley Road, 5209, San Diego, Calif. 92121). Shown in Table 3 in columns(A) and (B), actual % SA was calculated for proteins having therespective amino acid sequences set forth as SEQ ID NO:185 (TIC807_L11M)and SEQ ID NO:8 (TIC807_M2). The predicted % SA for residues of TIC807and TIC853 are listed in Table 3 in columns (A) and (C), respectively.Altogether, the % SA values reported in Table 3 are calculated as apercentage of the solvent-accessible surface area probed by a watermolecule over the maximal solvent accessible area in standard extendedconformation (Gly-XXX-Gly) for each residue in each position of theatomic structure. Table 3 aligns the residues of each protein by alignedresidues in a Clustal W alignment. % SA greater than 100 can occur whenmaximal solvent accessible area in standard extended conformation(Gly-XXX-Gly) for each residue is less than the actual solventaccessible area probed by a water molecule. % SA greater than 100 arereported in the table as 100%.

Combined engineering-testing-selecting approaches described hereinresulted in a number of principally relevant residues that accumulate ina surface patch ([2] of FIG. 2) of residues having a radius of about9.2-12.2 Angstroms around the Cb atom of P219 of SEQ ID NO:2 (TIC807):V10, I14, N22, N23, G24, I25, Q26, G27, F30, Q38, I39, D40, T41, I43,S193, T194, E195, H196, Y197, S198, H199, Y200, S201, G202, Y203, P204,I205, L206, T207, W208, I209, S210, Y216, S217, G218, P219, F220, M221,S222, W223, Y224, F225, N239, and V244 of SEQ ID NO:2 (TIC807). At leasthalf of these residues exhibit % SA values of greater or equal tofifteen (15).

TABLE 3 Relative % Solvent Accessability (SA) of Amino Acids of eHTP's &Scaffold Proteins. (A) (B) (C) TIC807_L11M TIC807_M2 TIC853 (SEQ ID NO:185) (SEQ ID NO: 8) (SEQ ID NO: 184) Position Position Position andamino Calculated and amino Calculated and amino Estimated acid % SA peracid % SA per acid % SA per residue residue residue residue residueresidue 2ALA 82.1# 2ALA 64.8# 2ALA 60.9# 3ILE 23.3  3ILE 28.9  3ILE24.3  4LEU 26.4  4LEU 31.9  4LEU 27.9  5ASP 26.0  5ASP 22.7  5ASP 29.9 6LEU 1.0 6LEU 3.4 6LEU 3.7 7LYS 25.1  7LYS 16.2  7LYS 17.1  8SER 46.4#8SER 37.6# 8SER 44.9# 9LEU 8.9 9LEU 5.2 9LEU 6.3 10VAL^(p1) 0.3 10VAL0.6 10VAL 0.0 11MET 25.0  11LEU 17.0  11LEU 16.6  12ASN* 49.8# 12ASN43.2# 12ASP 39.7# 13ALA 0.0 13ALA 0.0 13ALA 0.0 14ILE^(p1) 0.0 14ILE 0.014ILE 0.0 15ASN 23.7  15ASN 24.9  15ASN 19.2  16TYR 29.5  16TYR 47.1#16TYR 52.5# 17TRP 14.1  17TRP 18.2  17TRP 20.1  18GLY 4.3 18GLY 1.418GLY 1.0 19PRO 63.6# 19PRO 57.3# 19PRO 59.0# 20LYS 57.3# 20LYS 77.2#20LYS 100#   21ASN 36.3# 21ASN 28.4  21ASN 61.5# 22ASN^(p1) 16.9  22ASN10.1  22ASN 15.2  23ASN^(p1) 0.3 23ASN 0.8 23ASN 0.0 24GLY^(p1) 42.0#24GLY 43.2# 24GLY 43.3# 25ILE^(p1) 10.1  25ILE 13.8  25ILE 7.626GLN^(p1) 92.4# 26GLN 86.2# 26GLN 94.7# 27GLY^(p1) 62.0# 27GLY 73.9#27GLY 62.8# 28GLY 49.0# 28GLY 50.6# 28TYR 47.7# 29ASP 66.0# 29ASP 68.1#29ASN 80.7# 30PHE^(p1) 4.5 30PHE 4.1 30PHE 1.5 31GLY 37.2# 31GLY 41.4#31ASN 61.1# 32TYR 25.2  32TYR 25.3  32TYR 21.5  33PRO 70.7# 33PRO 76.0#33PRO 78.5# 34ILE 4.8 34ILE 5.5 34ILE 2.6 35SER 42.2# 35SER 29.1  35SER27.0  36GLU 54.2# 36GLU 47.2# 36GLU 50.2# 37LYS 81.0# 37LYS 79.5# 37ARG87.5# 38GLN^(p1) 12.8  38GLN 14.5# 38GLN 9.0 39ILE^(p1) 7.8 39ILE 7.939ILE 5.1 40ASP^(p1) 52.4# 40ASP 55.3# 40ASP 49.8# 41THR^(p1) 0.3 41THR0.0 41THR 0.2 42SER 53.1# 42SER 56.0# 42SER 53.0# 43ILE^(p1) 13.1  43ILE23.5  43ILE 25.1  44ILE 8.3 44ILE 12.0  44ILE 8.1 45THR 30.7  45THR37.8# 45THR 45.7# 46PHE* 20.0  46SER 43.7# 46SER 40.5# 47THR 48.1# 47THR45.2# 47THR 43.7# 48HIS 73.5# 48HIS 65.3# 48HIS 78.3# 49PRO 9.4 49PRO12.6  49SER 9.0 50ARG 58.7# 50ARG 53.7# 50ARG 61.5# 51LEU 13.7  51LEU8.1 51LEU 3.0 52ILE* 32.4  52ILE 31.5  52MET 43.7# 53PRO 22.2  53PRO26.5  53PRO 22.8  54TYR* 52.7# 54HIS 42.2# 54HIS 45.5# 55ASP 57.5# 55ASP59.2# 55ASP 55.5# 56LEU 15.0  56LEU 18.6  56LEU 15.1  57THR 62.0# 57THR73.2# 57THR 80.0# 58ILE 67.6# 58ILE 60.9# 58ILE 68.0# 59PRO 26.6  59PRO21.9  59PRO 20.7  60GLN 28.8  60GLN 20.7  60GLN 21.3  61ASN 71.9# 61ASN73.6# 61ASN 74.6# 62LEU 13.4  62LEU 11.7  62LEU 10.0  63GLU 62.2# 63GLU66.0# 63GLU 66.4# 64THR 51.2# 64THR 51.1# 64THR 49.0# 65ILE 46.1# 65ILE41.5# 65ILE 38.4# 66PHE 27.0  66PHE 26.3  66PHE 29.6  67THR 52.5# 67THR55.5# 67THR 62.2# 68THR* 31.6  68THR 35.8# 68THR 35.3  69THR 54.3# 69THR51.1# 69THR 50.3# 70GLN* 31.0  70GLN 36.5# 70GLN 34.3  71VAL 53.4# 71VAL53.2# 71VAL 53.1# 72LEU 7.9 72LEU 11.8  72LEU 8.1 73THR 40.5# 73THR44.0# 73THR 47.0# 74ASN 0.6 74ASN 0.0 74ASN 0.0 75ASN 69.8# 75ASN 63.2#75ASN 65.9# 76THR 36.7# 76THR 40.5# 76THR 44.0# 77ASP 80.2# 77ASP 63.0#77ASP 62.1# 78LEU 62.6# 78LEU 62.5# 78VAL 63.8# 79GLN 74.4# 79GLN 54.7#79GLN 43.5# 80GLN 33.3  80GLN 32.1  80GLN 40.1# 81SER 81.0# 81SER 68.6#81SER 75.2# 82GLN 19.0  82GLN 23.9  82GLN 24.6  83THR 62.7# 83THR 63.6#83THR 63.7# 84VAL 1.8 84VAL 0.9 84VAL 0.0 85SER 50.8# 85SER 55.7# 85SER54.0# 86PHE 7.2 86PHE 5.8 86PHE 4.0 87ALA* 58.5# 87ALA 61.9# 87SER 68.0#88LYS 30.5  88LYS 30.6  88LYS 32.6  89LYS 69.8# 89LYS 67.8# 89LYS 67.9#90THR 19.9  90THR 23.1  90THR 16.7  91THR 54.1# 91THR 55.1# 91THR 48.1# 92THR 1.8 92THR 1.8 92THR 0.0 93THR^(p2)* 40.3# 93THR 36.4# 93THR 29.4 94THR 0.0 94THR 0.6 94THR 0.2 95SER^(p2)* 14.4  95SER 15.7  95SER 18.1 96THR 5.5 96THR 1.5 96THR 0.0 97SER^(p2) 16.6  97SER 18.5  97SER 29.9 98THR 8.2 98THR 5.9 98THR 1.9 99THR 41.8# 99THR 46.4# 99THR 49.3# 100ASN37.7# 100ASN 34.1  100ASP 20.1  101GLY 1.0 101GLY 1.9 101GLY 0.0 102TRP3.6 102TRP 10.4  102TRP 6.6 103THR 8.1 103THR 8.1 103THR 3.9 104GLU 9.7104GLU 21.9  104GLU 14.8  105GLY* 35.3  105GLY 46.8# 105GLY 31.4  106GLY57.0# 106GLY 68.6# 106GLY 61.8# 107LYS 52.4# 107LYS 57.2# 107ARG 54.6#108ILE 61.8# 108ILE 63.5# 108ILE 67.1# 109SER 43.5# 109SER 47.9# 109SER47.7# 110ASP 83.1# 110ASP 83.5# 110ASP 65.7# 111THR 43.4# 111THR 41.3#111THR 39.9# 112LEU 26.7  112LEU 29.6  112LEU 31.3  113GLU 53.8# 113GLU64.1# 113GLU 62.5# 114GLU 34.8  114GLU 30.9  114GLU 32.6  115LYS 62.2#115LYS 55.2# 115ASN 54.6# 116VAL 6.4 116VAL 8.6 116VAL 10.4  117SER*46.6# 117SER 48.9# 117SER 51.2# 118VAL 0.9 118VAL 2.5 118VAL 1.3 119SER*20.2  119SER 23.4  119SER 23.7  120ILE 0.8 120ILE 0.3 120ILE 0.0 121PRO5.9 121PRO 10.7  121PRO 8.0 122PHE 0.2 122PHE 1.4 122PHE 0.3 123ILE19.1  123ILE 20.8  123ILE 18.3  124GLY 4.3 124GLY 6.2 124GLY 3.3 125GLU*59.7# 125GLU 56.2# 125ALA 57.8# 126GLY 50.0# 126GLY 52.5# 126GLY 49.6#127GLY 47.2# 127GLY 56.7# 127GLY 38.6# 128GLY* 34.7  128GLY 30.3  128ALA23.0  129LYS 68.8# 129LYS 73.9# 129LYS 78.4# 130ASN 16.2  130ASN 14.6 130ASN 10.1  131SER 78.2# 131SER 77.9# 131SER 80.3# 132THR 9.8 132THR10.3  132THR 12.3  133THR* 45.7# 133THR 42.0# 133THR 44.3# 134ILE* 1.1134ILE 0.8 134ILE 0.0 135GLU* 51.5# 135GLU 45.2# 135GLU 48.5# 136ALA 0.0136ALA 1.3 136ALA 2.4 137ASN* 18.1  137ASN 15.5  137ASN 15.6  138PHE*1.9 138PHE 0.9 138VAL 2.5 139ALA* 2.8 139ALA 6.3 139ALA 4.1 140HIS 2.3140HIS 2.1 140HIS 0.0 141ASN 5.3 141ASN 6.5 141ASN 2.8 142SER 5.4 142SER4.4 142SER 6.6 143SER 7.7 143SER 10.6  143SER 7.0 144THR 23.5  144THR17.3  144THR 16.6  145THR* 48.3# 145THR 52.7# 145THR 55.2# 146THR 50.2#146THR 49.7# 146THR 53.6# 147PHE^(p2)* 49.9# 147PHE 61.6# 147SER 51.7#148GLN* 12.9  148GLN 17.8  148GLN 18.4  149GLN^(p2)* 59.5# 149GLN 65.1#149GLN 69.1# 150ALA* 6.9 150ALA 8.7 150ALA 9.1 151SER^(p2)* 51.0# 151SER51.7# 151SER 57.9# 152THR 9.9 152THR 8.7 152THR 12.3  153ASP* 83.5#153ASP 84.5# 153GLU 63.3# 154ILE 11.2  154ILE 6.1 154ILE 6.3 155GLU*49.5# 155GLU 63.9# 155GLU 49.7# 156TRP 1.7 156TRP 3.8 156TRP 1.8 157ASN*59.1# 157ASN 59.1# 157ASN 53.4# 158ILE* 13.1  158ILE 5.9 158ILE 0.8159SER* 60.2# 159SER 52.9# 159SER 52.2# 160GLN 29.2  160GLN 19.3  160GLN9.3 161PRO 54.0# 161PRO 63.6# 161PRO 62.6# 162VAL 0.6 162VAL 4.0 162VAL2.4 163LEU 53.8# 163LEU 56.6# 163LEU 64.5# 164VAL 0.0 164VAL 0.0 164VAL0.0 165PRO 22.8  165PRO 22.1  165PRO 26.9  166PRO 30.7  166PRO 36.1#166PRO 39.7# 167SER* 31.0  167ARG 32.8  167ARG 36.7# 168LYS 18.2  168LYS18.5  168LYS 19.9  169GLN 17.4  169GLN 15.1  169GLN 10.7  170VAL 0.0170VAL 0.0 170VAL 0.0 171VAL 13.2  171VAL 13.8  171VAL 12.2  172ALA 0.0172ALA 0.0 172ALA 0.0 173THR 9.8 173THR 9.2 173THR 6.5 174LEU 1.3 174LEU2.6 174LEU 0.2 175VAL* 17.2  175VAL 17.8  175VAL 13.4  176ILE 0.0 176ILE0.0 176ILE 0.0 177MET* 7.0 177MET 7.7 177MET 17.3  178GLY 1.6 178GLY 0.5178GLY 0.0 179GLY 15.9  179GLY 22.2  179GLY 16.5  180ASN^(p2)* 60.0#180ASN 60.1# 180ASP 44.9# 181PHE 0.7 181PHE 2.8 181PHE 1.8 182THR^(p2)*50.6# 182THR 44.3# 182THR 40.8# 183ILE 0.0 183ILE 1.1 183VAL 0.0 184PRO36.6# 184PRO 34.2  184PRO 34.5  185MET 4.4 185MET 2.1 185MET 1.8 186ASP52.4# 186ASP 23.5  186ASP 20.3  187LEU* 0.8 187LEU 0.0 187LEU 0.0 188MET25.9  188MET 12.7  188ILE 24.9  189THR 1.4 189THR 2.9 189THR 0.5 190THR26.1  190THR 26.2  190THR 24.1  191ILE 4.0 191ILE 6.2 191ILE 1.8 192ASP25.9  192ASP 29.2  192ASP 21.4  193SER^(p1) 7.4 193SER 7.7 193SER 2.7194THR^(p1) 66.2# 194THR 60.2# 194THR 59.9# 195GLU^(p1) 38.5# 195GLU35.0  195GLN 35.5# 196HIS* 37.7# — 100    — 100#   197TYR* 32.2  —100#   — 100#   198SER* 35.5# — 100#   — 100#   199HIS^(p1)* 64.3#196HIS 55.5# 196HIS 51.7# 200TYR^(p1)* 85.3# 197TYR 56.2# 197PHE 45.8#201SER^(p1)* 50.3# 198SER 68.3# 198THR 64.5# 202GLY^(p1) 32.8  199GLY50.0# 199GLY 51.1# 203TYR^(p1) 21.5  200TYR 22.6  200TYR 26.0 204PRO^(p1) 1.4 201PRO 1.0 201PRO 1.0 205ILE^(p1) 1.1 202ILE 0.3 202ILE0.0 206LEU^(p1) 1.8 203LEU 2.6 203LEU 0.0 207THR^(p1) 0.0 204THR 0.0204THR 0.0 208TRP^(p1)* 38.8# 205TRP 35.6# 205TRP 22.5  209ILE^(p1) 0.0206ILE 0.0 206ILE 0.0 210SER^(p1) 22.5  207SER 20.1  207GLU 17.0  211SER3.1 208SER 3.4 208ASN 4.6 212PRO 56.5# 209PRO 58.4# 209PRO 56.2# 213ASP68.0# 210ASP 55.2# 210GLU 60.5# 214ASN 65.5# 211ASN 66.4# 211HIS 64.4#215SER 67.2# 212SER 74.1# 212ASN 74.2# 216TYR^(p1) 42.5# 213TYR 39.8#213VAL 29.4  217SER^(p1)* 43.2# 214ASN 46.3# 214ARG 57.1# 218GLY^(p1)1.2 215GLY 4.1 215GLY 6.1 219PRO^(p1)* 14.4  216PRO 14.7  216ARG 33.7 220PHE^(p1) 0.0 217PHE 0.0 217PHE 0.0 221MET^(p1) 15.2  218MET 16.1 218LEU 8.4 222SER^(p1) 3.3 219SER 3.3 219SER 0.0 223TRP^(p1)* 35.6#220TRP 34.3  220TRP 42.5# 224TYR^(p1) 13.5  221TYR 15.9  221PHE 11.3 225PHE^(p1) 0.9 222PHE 1.4 222PHE 0.0 226ALA 15.9  223ALA 13.1  223ALA7.8 227ASN 40.7# 224ASN 41.9# 224ASN 43.2# 228TRP 9.0 225TRP 8.9 225TRP7.3 229PRO 56.3# 226PRO 61.5# 226PRO 65.5# 230ASN 67.6# 227ASN 67.3#227ASN 67.3# 23ILEU 21.1  228LEU 16.1  228LEU 16.1  232PRO 23.7  229PRO23.0  229PRO 23.6  233SER 97.0# 230SER 95.8# 230SER 88.1# 234GLY 23.0 231GLY 19.5  231GLU 13.0  235PHE* 8.3 232PHE 9.0 232PHE 6.1 236GLY 26.1 233GLY 18.3  233GLY 28.5  237PRO 72.6# 234PRO 70.8# 234SER 81.9# 238LEU27.7  235LEU 28.1  235LEU 25.9  239ASN* 33.2  236ASN 26.5  236ASN 42.2#240SER 100#   237SER 100#   237SER 100#   241ASP* 62.2# 238ASP 61.7#238ASP 55.2# 242ASN 15.1  239ASN 20.8  239ASN 21.7  243THR* 3.3 240THR2.4 240THR 2.6 244VAL* 1.8 241VAL 3.1 241ILE 0.0 245THR* 19.0  242THR23.8  242THR 30.1  246TYR* 8.6 243TYR 4.8 243TYR 0.4 247THR* 36.8#244THR 40.8# 244LYS 58.8# 248GLY 2.5 245GLY 1.4 245GLY 0.0 249SER* 20.7 246SER 23.9  246SER 27.0  250VAL* 6.0 247VAL 1.4 247VAL 0.0 251VAL^(p2)*32.6  248VAL 30.0  248VAL 29.7  252SER* 0.0 249SER 0.0 249SER 0.0253GLN^(p2) 40.2# 250GLN 37.6# 250ARG 51.3# 254VAL 1.2 251VAL 1.5 251ILE2.7 255SER^(p2) 35.3  252SER 37.3# 252SER 43.7# 256ALA 6.1 253ALA 2.4253ALA 2.1 257GLY 4.1 254GLY 6.1 254GLY 1.2 258VAL 0.3 255VAL 0.0 255VAL0.0 259TYR 2.2 256TYR 1.1 256TYR 1.0 260ALA 0.7 257ALA 0.7 257ALA 0.0261THR 9.2 258THR 9.3 258THR 5.0 262VAL 3.6 259VAL 1.5 259VAL 0.2 263ARG26.6  260ARG 29.8  260ARG 26.9  264PHE 0.5 261PHE 3.8 261PHE 1.5 265ASP6.9 262ASP 7.2 262ASP 10.8  266GLN 5.6 263GLN 5.8 263GLN 2.6 267TYR16.1  264TYR 14.5  264TYR 12.4  268ASP 29.8  265ASP 31.4  265ALA 19.3 269ILE 25.4  266ILE 18.2  266ILE 14.5  270HIS 85.5# 267HIS 72.2# 267ASN92.0# 271ASN 43.4# 268ASN 46.9# 268ASN 64.0# 272LEU 40.3# 269LEU 43.1#269LEU 39.8# 273ARG* 86.3# 270ARG 63.1# 270ARG 84.4# 274THR* 52.0#271THR 66.1# 271THR 76.8# 275ILE* 41.0# 272ILE 37.9# 272ILE 32.4  276GLU47.9# 273GLU 50.1# 273GLU 53.0# 277LYS 49.8# 274LYS 47.2# 274LYS 70.2#278THR 46.3# 275THR 51.2# 275THR 53.7# 279TRP 25.2  276TRP 25.0  276TRP33.4  280TYR 35.5  277TYR 30.7  277TYR 21.3  281ALA 6.6 278ALA 7.9278ALA 4.4 282ARG* 77.6# 279ARG 80.6# 279ARG 86.1# 283HIS 45.2# 280HIS36.6# 280HIS 35.8# 284ALA 0.8 281ALA 0.8 281GLY 0.6 285THR 14.7  282THR8.6 282THR 2.0 286LEU 3.6 283LEU 5.9 283LEU 2.3 287HIS* 8.4 284HIS 16.5 284HIS 11.9  288ASN 40.4# 285ASN 43.9# 285ASN 38.7# 289GLY 61.0# 286GLY53.5# 286GLY 61.1# 290LYS 61.7# 287LYS 61.6# 287LYS 73.8# 291LYS 68.4#288LYS 66.2# 288LYS 51.9# 292ILE 19.2  289ILE 19.5  289ILE 21.0  293SER*40.3# 290SER 47.9# 290SER 45.7# 294ILE 3.4 291ILE 4.8 291ILE 5.1 295ASN*29.3  292ASN 21.9  292ASN 18.0  296ASN 38.2# 293ASN 40.4# 293ASN 37.4#297VAL 1.3 294VAL 1.4 294VAL 0.7 298THR 10.1  295THR  9.5# 295THR 4.3299GLU* 77.1# 296GLU 72.7# 296GLU 68.8# 300MET* 48.6# 297MET 46.4#297MET 42.8# 301ALA 65.3# 298ALA 54.1# 298ALA 60.4# 302PRO 66.0# 299PRO73.0# 299PRO 77.8# 303THR* 83.7# 300THR 85.8# 300THR 94.1# 304SER 77.4#301SER 76.1# 301SER 84.9# 305PRO* 81.1# 302PRO 65.7# 302PRO 83.4#306ILE* 78.1# 303ILE 81.6# 303ILE 91.3# 307LYS 81.9# 304LYS 99.3# 304GLU100#   308THR* 89.4# 305THR 100#   305ARG 100#   309ASN 100#   306ASN100#   306ASN 100#   P1 designates an amino acid in surface patch [1] ofFIG. 2. P2 designates an amino acid in surface patch [1] of FIG. 2.*designates one of the 72 principally relevant amino acids describedherein (see FIG. 2). Shown are residues of TIC807_L11M, TIC807_M2, andTIC853 aligned by Clustal W. Numbers marked with # represent % SA of atleast about 36%.Receptor Binding

A surface patch ([1] of FIG. 2) of residues having % SA values greaterthan 36% or within about 3 residues of a residue having % SA greaterthan 36% in a radius of about 9.2-12.2 Angstroms from the Cb atom of S95of SEQ ID NO:2 (TIC807) was identified as a region comprising residuesof a TIC807 protein that can be substituted to provide for eHTP's thatexhibit enhanced Lygus inhibitory spectrum and/or improved Lygusinhibitory activity, This surface patch region may be associated withtarget insect receptor binding activity; and, includes residues T93,S95, S97, F147, Q149, S151, N180, T182, V251, Q253, and S255 of SEQ IDNO:2 (TIC807). eHTP's can include, but are not limited to, one or moresubstitutions of surface patch 1 amino acid residues such as S95A,F147A, Q149E, and/or, V251A.

The combined engineering-testing-selecting approaches described hereinidentified residues located in surface patch 1 that can provide foreHTP's when substituted or otherwise modified. These residues may beimportant for productive binding of eHTP's to receptors in Lygus insectgut to provide for enhanced Lygus inhibitory spectrum and/or improvedLygus inhibitory activity when compared to TIC807. Modifications of thesurface patch 1 amino acid residues that can provide for eHTP's includesubstitutions that provide aromatic groups and/or hydrogen-bondinggroups which favoring binding to sugar groups found on glycosylatedreceptors of insects.

Membrane Binding

Certain amino acid residues located in beta-sheet regions of the proteinwere identified from the atomic structure of TIC807 and were substitutedwith aromatic residues. More specifically, amino acids L78, I123, H270,R273, I275 of the folded TIC807 beta sheet regions were substituted withPhenyalanine, Tyrosine, or Tryptophan. Aromatic amino acid substitutionsof R273 and I275 were amongst those residues that provided for anenhanced Lygus inhibitory spectrum and/or improved Lygus inhibitoryactivity (See Table 4, data for SEQ ID NOs:32, 34, 68, 92, and 122).Amino acid side chains of residues in these positions may be likely tointeract with the membrane of target insects.

Proteolytic Activation Sites

Glycine residues generally thought to be involved in proteolysis weresubstituted with Serines to alter proteolytic cleavage dynamics. Thepresence of a glycine residue in a loop region can impart moreflexibility and therefore susceptibility to proteolysis, which caneither increase insect inhibitory activity or decrease insect inhibitoryactivity. Residues in structurally identified loop regions weresubstituted with a glycine residue, and no improvements were observed.Positions in loops that were already glycines, (e.g. G18, G24, G27) weresubstituted with a serine, a small residue in an attempt to reduceproteolytic susceptibility, and no improvements were observed.

Combined Structure Design Approaches

The atomic structure of TIC807 (SEQ ID NO:2) was used to identify loopregions for library mutagenesis followed by testing of the engineeredvariants. A loop at amino acid positions 211-216 of SEQ ID NO:2 (TIC807)was library-mutagenized and tested. Consecutive loops in close proximityat amino acid positions 75-83, 161-167, and 267-276 of SEQ ID NO:2(TIC807) was library-mutagenized and tested.

Analysis of the atomic structure of TIC807 suggests that a structuralloop resides at residues 113-138 of SEQ ID NO:2, and variants wereengineered to stabilize and destabilize the loop.

In another region spanning two beta-strands connected by a short loop,the two beta-strands exhibited an alternating pattern of hydrophobic andhydrophilic amino acid residues at positions 116 to 121 and at positions133 to 138 relative to SEQ ID NO:2, characteristic of pore-formingloops. An expression library was engineered to modify both beta-strandsegments replacing residues V116, V118, and I120 with respectivecombinations 116V/Y/L/H/F/D, 118V/Y/L/H/F/D, and 120I/D/F/H/L/N/V/Y fora total of 288 possible variants in the library. This procedure wasrepeated for: residues S117, S119, and P121 with respective combinations117S/A/D/E/G/K/N/R/T, 119S/A/D/E/G/K/N/R/T, and 121P/S/T for 243potential variants; residues I133, A135, and F137 with respectivecombinations 133I/D/F/N/V/Y, 135A/D/F/H/L/V/Y, and 137F/D/H/L/V/Y for252 possible variants; and residues T134, E136, and N138 with respectivecombinations 134T/A/D/E/G/K/N/R/S, 136E/A/D/G/K/N/R/S/T, and138N/A/D/G/S/T for 486 possible variants. An enhanced Lygus inhibitoryspectrum and/or improved Lygus inhibitory activity was associated withcertain of these substitutions as shown in Table 4.

Structure-Function Relationship

Altogether, more than 2000 clones (including mixed library clones)expressing variants of TIC807 were tested for enhanced Lygus inhibitoryspectrum and/or improved Lygus inhibitory activity against Lygus spp.compared to TIC807. Semi-random modifications, directed modifications,and predictive structure-function modifications, including structuremodeling, receptor binding potential, metal binding potential,oligomerization potential, uniformity of surface charge distribution,pore formation potential, ion channel function, and identification ofsurface exposed patches to with an objective of identifying eHTP's withan enhanced Lygus inhibitory spectrum and/or improved Lygus inhibitoryactivity compared to TIC807. These clones were expressed for bioassaytesting.

Example 3: Protein Expression and Purification of TIC807, IncludingVariants and Fragments

Control protein TIC807 is a protein of 309 amino acids in length thatcan be expressed in crystalline form in Bacillus thuringiensis (Bt) oraggregate form in E. coli. Test variants thereof were recombinantlyexpressed in Bt. An expression characteristic of TIC807 and variants ofTIC807 is the predominant crystalline and aggregate forms extracted fromBt and E. coli cells, respectively. To test for Lygus bioactivity, testand control samples were made suitable for Lygus bioassay bysolubilizing samples in 25 mM Sodium Carbonate buffer and removingunsolubilized materials by centrifugation. The amount of protein in testand control samples were measured using total protein methods, e.g.s, aBradford assay, an ELISA method, or similar. Gel electrophoresis wasused to determine the purity and stock concentration of the solubilizedrecombinant protein. C-terminal HIS-tagged TIC807 protein was engineeredto facilitate detection, purification, and quantification of largeamounts of TIC807 control protein. C-terminal HIS-tagged TIC807 andun-tagged TIC807 test samples were separately assayed and confirmed tohave equivalent activity against Lygus (see Examples 4, 5, and 6).

Site-directed amino acid substitutions were made to TIC807_M13 (SEQ IDNO:34) to elevate expression of a soluble form. Inventors postulate thatmore readily soluble variants of the proteins of the present inventioncan facilitate expression and purification, e.g., expressed in E. colihost cells; and can increase insect inhibitory efficacy when expressedin plant host cells. Recombinant DNA constructs encoding TIC807_M13 (SEQID NO:34) were engineered three different ways to reflect threedifferent variants: Relative to TIC807_M13, the modifications were forVariant #1: I58K and P59K, for Variant #2: S198K and G199K, and forVariant #3: S246R, V248E, and Q250R. Relative to TIC807 (SEQ ID NO:2),the modifications can be alternatively described as follows for Variant#1: I58K and P59K, for Variant #2: S201K and G202K, and for Variant #3:S249R, V251E, and Q253R; this positional difference is congruent due toa contiguous triple deletion of SEQ ID NO:2 (TIC807) in residue range196-201 that is reflected in TIC807_M13 (SEQ ID NO:34). The fourengineered recombinant DNA constructs were each cloned and expressed inE. coli. The soluble fraction from the four E. coli preparations wereevaluated by coomassie-stained SDS-PAGE, which showed that TIC807_M13(SEQ ID NO:34) was not detectable in the soluble fraction; but, incontrast, Variant #s 1, 2, and 3 were soluble. Similar amino acidsubstitutions either singly or in combination are made to proteins ofthe present invention to elevate their solubility in non-Bt or planthost cells. Recombinant DNA constructs were engineered to encode for andexpress TIC807_M13 variant #3 (renamed TIC807_M14; nucleotide SEQ IDNO:203 and amino acid SEQ ID NO:204). Prepared E. coli lysate wasclarified, and the recombinant protein purified and enriched-for on aseries of columns, including ion-exchange and gel filtration methods.Pooled protein fractions were quantified and determined to be activeagainst Lygus insects (See Example 4, Table 4B).

Proteins of the present invention, including but not limited to proteinshaving the amino acid sequence as set forth as SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, or SEQ ID NO:36, are engineered to elevateexpression of a soluble form when expressed in a host cell, e.g.,expressed in Bt, E. coli, or in a plant cell or in a compartment of aplant cell. Engineering includes substituting a lysine amino acidresidue at one or more of the following positions 58, 59, 198, 199, 201,or 202; or, a Glutamic acid at one or more of the following positions198, 248, or 301; or, an Arginine at one or more of the followingpositions 246, 250, or 253.

The C-terminal region protrudes away from the monomeric core of theprotein (See FIG. 2). A recombinant DNA construct was engineered toencode for and express a protein having the amino acid sequence of SEQID NO:202, which is a protein fragment (amino acids 1 to 301) ofTIC807_M8 (SEQ ID NO:16); and, the expressed protein was purified,quantified, and determined active against Lygus insects (See Example 4,Table 4B). Recombinant DNA constructs were designed to encode for andexpress TIC807 fragments exhibiting varying truncations off of theC-terminus end of the proteins of the present invention at therespective TIC807 positions A281, G289, S293, A301, and S304. Proteinfragments are engineered to encode for and express proteins having theamino acid sequences set forth as SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, and SEQ ID NO:36; and, the expressed proteinfragments are used as test samples against Lygus insects.

Example 4: Hemipteran Activity of Engineered Proteins

This example illustrates eHTP's to have improved insecticidal activityor enhanced insecticidal specificity against Hemipteran insects whenprovided in the diet of Hemipteran insects, including but not limited tomembers of the Heteroptera miridae, including the genus Lygus, e.g.,Lygus hesperus and Lygus lineolaris, and the family Cicadellidae,including the genus Amrasca, e.g. Amrasca devastans, and Empoasca, e.g.Empoasca fabae. This example with Table 4B illustrates the feeding assayused to determine the enhanced Lygus inhibitory spectrum and/or improvedLygus inhibitory activity of a Bt expressed recombinant proteins of thepresent invention against both Lygus hesperus and Lygus lineolaris.Proteins expressed in recombinant bacterium host cells were solubilizedin carbonate buffer and analyzed by SDS polyacrylamide gelelectrophoresis (SDS-PAGE); and, protein concentrations determined bydensitometry using bovine serum albumin (BSA) as a standard. Proteinstock (2×) prepared this way were mixed with diet for feeding assays.

Feeding assays with the Hemipteran species Lygus hesperus and Lyguslineolaris were based on a 96 well micro-titer plate format with Lygusdiet encapsulated between stretched Parafilm® and Mylar sheets.Artificial diet was obtained from Bio-Serv® (Bio-Serv® Diet F9644B,Frenchtown, N.J.). Autoclaved, boiling water (518 mL) was combined with156.3 grams of Bio-Serv® diet F9644B in a surface-sterilized blender.The contents of four surface-sterilized chicken eggs were added and themixture blended until smooth, then adjusted to one liter total volumeand allowed to cool to room temperature, this being the 2× diet. Testsamples were prepared by mixing in a 1:1 ratio of 2× diet and 2× sample.A sheet of Parafilm® (Pechiney Plastic Packing, Chicago, Ill.) wasplaced over a vacuum manifold designed for 96-well format (AnalyticalResearch Systems, Gainesville, Fla.) and a vacuum of approximately −20millimeters mercury was applied, sufficient to cause extrusion of theParafilm® into the wells. Twenty to forty microliters of test samplewere added to the Parafilm® extrusions. A sheet of Mylar film (Clear LamPackaging, Inc., Elk Grove Village, Ill.) was placed over the samplefilled Parafilm® extrusions and sealed with a tacking iron (BienfangSealector II, Hunt Corporation, Philadelphia, Pa.), thus forming dietfilled Parafilm® sachets. These Parafilm® sachets were positioned over aflat-bottom 96-well plate containing Lygus eggs suspended in a diluteagarose solution. Upon hatching, Lygus nymphs feed on the diet bypiercing the diet filled Parafilm® sachets. Alternatively, newly hatchedLygus nymphs instead of eggs were manually infested into each well.Stunting and mortality scores were determined on day 5 and compared tocontrols. Data were analyzed using JMP4 statistical software. For eachprotein at a test concentration, three populations of eight nymphs weresubjected to this bioassay, and mortality scores reported in Table 4B.

For LC50 determinations listed in Table 1 and Table 4B, proteins werepresented to newly hatched Lygus nymphs at 8-10 concentrations and thenymphs allowed to feed for 5 days before scoring for mortality over thedose range. For each concentration, three populations of eight nymphswere subjected to this bioassay, and all LC50 determinations in Table 1and Table 4B were repeated at least once.

For LC50 estimations, proteins were presented to newly hatched Lyguslineolaris nymphs at 4 concentrations and the nymphs allowed to feed for5 days before scoring for mortality over the dose range. Lyguslineolaris LC50 estimations were performed on TIC807 and TIC807_M2because significantly large amounts of these proteins in excess of 1000μg/mL have not been possible to provide in Lygus diet in order tocomplete the high range of toxicity dose response to Lygus lineolaris;and therefore, an LC50 value was not determined for TIC807 or TIC807_M2.Instead, a 4-dose LC50 estimation in the low range was performed, andreported in Table 1 and Table 4B. The estimated Lygus lineolaris LC50for TIC807_M14 is 4.4 μg/mL. For each concentration, three populationsof eight nymphs were subjected to this bioassay.

This example with Tables 4A and 4B illustrate the feeding assay used todetermine the enhanced inhibitory spectrum and/or improved inhibitoryactivity of a Bt expressed recombinant protein disclosed herein againstAmrasca devastans. TIC807 variants with improved insecticidal activityor enhanced insecticidal specificity against Lygus hesperus and Lyguslineolaris exhibit improved insecticidal activity against Amrascadevastans.

TIC807, and TIC807-M13 were dissolved in 25 mM sodium carbonate buffer,pH 10. Amrasca devastans eggs were collected on Okra leaf and incubatedin a petriplate containing 2% agar. Upon hatching the neonates were usedfor bioassays using the diluted (1:5) Lygus diet. The proteins and dietwere mixed at equal proportion (bringing final concentration of proteinto 500 μg/mL) and dispensed into test arena. Untreated control wasprepared by mixing the buffer with the diet. Individual neonates wereinfested into the test arena, the assays were incubated at 25° C., 60%RH. Twenty neonate nymphs were tested for each concentration, proteinand in 2 replicates. A control was maintained with 25 mM SodiumCarbonate buffer, pH 10, in 1:5 diluted Lygus diet. Mortality of theinsects was determined on the fifth day. Mortality values werecalculated by the following formula: (% mortality in treatment−%mortality in control)/(100−% mortality in control)×100. Table 4Atabulates Amrasca activity for TIC807 and TIC807_M13 at 5 differentconcentrations.

TABLE 4A TIC807 and TIC807_M13 Percent Mortality Directed to Amrascaspecies SEQ Mortality (%) ID 500 166.66 55.55 18.51 6.17 NO: ProteinName μg/mL μg/mL μg/mL μg/mL μg/mL 2 TIC807 100% 55.88% 17.64% 0 0 34TIC807_M13 100% 88.23% 73.52% 44.11% 26.47%

LC50 values were determined for TIC807 and TIC807_M13 in a separatetest. SEQ ID NO:2 (TIC807) exhibited a LC50 value of 116.79 μg/mL andLC90 of 437.27 μg/mL. SEQ ID NO:34 (TIC807_M13) exhibited a LC50 valueof 7.59 μg/mL and LC90 value of 239.8 μg/mL.

A feeding assay as described for Amrasca devastans is used to testeHTP's for improved insecticidal activity and/or enhanced insecticidalspecificity against Empoasca fabae. TIC807 variants with improvedinsecticidal activity or enhanced insecticidal specificity against Lygushesperus and Lygus lineolaris exhibit improved insecticidal activityagainst Empoasca fabae.

The LC50 values of Cry51Aa1 (SEQ ID NO:182), for TIC807 (SEQ ID NO:2),TIC807_M2 (SEQ ID NO:8), TIC807_M10 (SEQ ID NO:30) and TIC807-M13 (SEQID NO:34) against Lygus hesperus and Lygus lineolaris were determined inone testset. TIC807_M2, TIC807_M10 and TIC807_M12 exhibit improved LC50values compared to Cry51Aa1.

It should be apparent to those skilled in the art that variations tothis procedure can exist that should not affect results.

TABLE 4B Iterative engineering-testing-selecting of eHTP's against Lygusspp. resulted in 267 proteins with enhanced Lygus inhibitory spectrumand/or improved Lygus inhibitory activity against Lygus spp. compared toTIC807. Lygus hesperus Lygus lineolaris Amino acid % % differencescompared to TIC807 parent protein Mortality Mortality Amino acid Aminoacid fold at about fold at about SEQ Amino acid difference(s)difference(s) LC50 increased 1-3 LC50 increased 100 ID difference(s)relative to in the in the value toxicity μg/mL value toxicity μg/mL NO:Protein Name SEQ ID NO: 2 (TIC807) first patch second patch (μg/mL)(LC50) protein* (μg/mL) (LC50) protein* 2 TIC807 Parent Parent Parent 731  0 >223 1  0 6 TIC807_M1 F147A F147A None 23 3 ND 100 at least ND 2 8TIC807_M2 F46S, Y54H, S167R, none S217N, then a 6.0 12 39 >223 — 14S217N, then a contiguous triple contiguous triple deletion in residuedeletion in residue range 196-201 range 196-201 10 TIC807_M3 F46S, Y54H,T93A, T93A S217N, then a 2.9 25 43 ND — ND S167R, S217N, then contiguoustriple a contiguous triple deletion in residue deletion in residue range196-201 range 196-201 12 TIC807_M4 F46S, Y54H, S95A, S95A S217N, then a2.4 30 20 ND — ND S167R, S217N, then a contiguous triple contiguoustriple deletion in residue deletion in residue range 196-201 range196-201 14 TIC807_M5 F46S, Y54H, F147A, F147A S217N, then a 1.1 66 34 ND— ND S167R, S217N, then a contiguous triple contiguous triple deletionin residue deletion in residue range 196-201 range 196-201 16 TIC807_M8F46S, Y54H, S95A, S95A, S217N, then a 0.8 91 ND 223 at least ND F147A,S167R, S217N, F147A contiguous triple 1 then a contiguous tripledeletion in residue deletion in residue range 196-201 range 196-201 18TIC807_M6 F46S, Y54H, T93A, T93A, S217N, then a 1.5 50 ND ND — ND F147A,S167R, S217N, F147A contiguous triple then a contiguous triple deletionin residue deletion in residue range 196-201 range 196-201 20 TIC807_M7F46S, Y54H, Q149E, Q149E S217N, then a 1.4 52 62 ND — 49 S167R, S217N,then a contiguous triple contiguous triple deletion in residue deletionin residue range 196-201 range 196-201 28 TIC807_M9 F46S, Y54H, S95A,S95A, P219R, then a 9.9 7 ND 8.3 at least ND F147A, S167R, P219R, F147Acontiguous triple 27 then a contiguous triple deletion in residuedeletion in residue range 196-201 range 196-201 30 TIC807_M10 F46S,Y54H, S95A, S95A, P219R, then a 0.6 122 ND 4.8 at least ND F147A, S167R,P219R, F147A, contiguous triple 46 V251A, then a V251A deletion inresidue contiguous triple range 196-201 deletion in residue range196-201 32 TIC807_M11 F46S, Y54H, S95A, S95A, P219R, then a 1.4 54 ND5.9 at least ND F147A, S167R, P219R, F147A contiguous triple 38 R273W,then a deletion in residue contiguous triple range 196-201 deletion inresidue range 196-201 34 TIC807_M13 F46S, Y54H, S95A, S95A, P219R, thena 0.3 243 ND 0.85 at least ND F147S, Q149E, S167R, F147A, contiguoustriple 262 P219R, R273W, then a Q149E deletion in residue contiguoustriple range 196-201 deletion in residue range 196-201 36 TIC807_M12F46S, Y54H, S95A, S95A, P219R, then a 0.4 182 ND 1.2 at least ND F147A,S167R, P219R, F147A, contiguous triple 186 N239A, V251A, then a V251Adeletion in residue contiguous triple range 196-201 deletion in residuerange 196-201 37 TIC807_37 TIC807_HYS_deletion none a contiguous 22.4 3ND ND — ND triple deletion in HYSHYS residues (positions 196-201) 38TIC807_38 F46S, Y54H, F138V, none S217N, then a ND — 41 ND — 44 S167R,S217N, then a contiguous triple contiguous triple deletion in residuedeletion in residue range 196-201 range 196-201 39 TIC807_39 F46S, Y54H,S167R, none S217N, then a ND — 57 ND — 31 S217N, H287F, then acontiguous triple contiguous triple deletion in residue deletion inresidue range 196-201 range 196-201 40 TIC807_40 F46S, I52M, Y54H, noneS217N, then a ND — 52 ND — 36 S167R, S217N, then a contiguous triplecontiguous triple deletion in residue deletion in residue range 196-201range 196-201 41 TIC807_41 N12D, F46S, Y54H, S217N, then a ND — 52 ND —31 S167R, S217N, then a none contiguous triple contiguous tripledeletion in residue deletion in residue range 196-201 range 196-201 42TIC807_42 F46S, Y54H, S167R, N180D S217N, then a ND — 63 ND — 50 N180D,S217N, then a contiguous triple contiguous triple deletion in residuedeletion in residue range 196-201 range 196-201 43 TIC807_43 F46S, Y54H,S167R, none a contiguous ND — 46 ND — 87 then a contiguous triple tripledeletion deletion in residue in HYSHYS range 196-201 residues (positions196-201) 44 TIC807_44 F46S, Y54H, S167R, none S217N, P219R, ND — 30 ND —94 S217N, P219R, then a then a contiguous contiguous triple tripledeletion in deletion in residue residue range range 196-201 196-201 45TIC807_45 F46S, Y54H, S159T, none S217N, then a ND — 67 ND — 21 S167R,S217N, then a contiguous triple contiguous triple deletion in residuedeletion in residue range 196-201 range 196-201 46 TIC807_46 F46S, Y54H,S167R, none S217N, then a ND — 52 ND — 31 S217N, 1247K, then acontiguous triple contiguous triple deletion in residue deletion inresidue range 196-201 range 196-201 47 TIC807_47 F46S, Y54H, S167R, noneS217N, then a ND — 59 ND — 31 S217N, V244I, then a contiguous triplecontiguous triple deletion in residue deletion in residue range 196-201range 196-201 48 TIC807_48 F46S, Y54H, S167R, none S217N, then a ND — 58ND — 34 S217N, V244I, T247K, contiguous triple then a contiguous tripledeletion in residue deletion in residue range 196-201 range 196-201 49TIC807_49 F46S, Y54H, S167R, none S217N, W223Y, ND — 17 ND — 13 S217N,W223Y, then a then a contiguous contiguous triple triple deletiondeletion in residue in residue range 196-201 range 196-201 50 TIC807_50F46S, Y54H, S167R, none S217N, then a ND — 48 ND — 19 S217N, Y246F, thencontiguous triple contiguous triple deletion in residue deletion inresidue range 196-201 range 196-201 196-201 51 TIC807_51 F46S, Y54H,F147A, F147A S217N, then a ND — 77 ND — ND G128A, S167R, S217N,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 52 TIC807_52 F46S, Y54H, F147A,F147A S217N, then a ND — 73 ND — ND S167R, S217N, M300A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 53 TIC807_53 F46S, Y54H, F147A, F147A S217N,then a ND — 73 ND — ND S167R, S217N, S293A, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 54 TIC807_54 F46S, Y54H, F147A, F147A S217N, then a ND —67 ND — ND S167R, S217N, H287A, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 55 TIC807_55 F46S, Y54H, F147A, F147A S217N, then a ND — 65 ND —ND S167R, S217N, T274A, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 56TIC807_56 F46S, Y54H, F147A, F147A S217N, then a ND — 64 ND — ND S167R,S217N, R282A, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 57 TIC807_57F46S, Y54H, T93A, T93A S217N, then a ND — 63 ND — ND S167R, S217N,T308A, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 — 58 TIC807_58 F46S,Y54H, Q70A, T93A S217N, then a ND 61 ND — ND T93A, S167R, S217N,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 59 TIC807_59 F46S, Y54H, E125A,F147A then a S217N, ND — 61 ND — ND F147A, S167R, S217N, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 60 TIC807_60 F46S, Y54H, F147A, F147A S217N,then a ND — 61 ND — ND S167R, S217N, 1247A, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 61 TIC807_61 F46S, Y54H, T93A, T93A S217N, then a ND —59 ND — ND S167R, S217N, P305A, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 62 TIC807_62 F46S, Y54H, F147A, F147A S217N, then a ND — 57 ND —ND S167R, S217N, I306A, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 63TIC807_63 F46S, Y54H, T93A, T93A S217N, then a ND — 56 ND — ND S167R,S217N, R282A, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 64 TIC807_64F46S, Y54H, T93A, T93A S217N, then a ND — 55 ND — ND S167R, S217N,T308A, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 65 TIC807_65 F46S, Y54H,T93A, T93A S217N, then a ND — 55 ND — ND S167R, S217N, M300A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 66 TIC807_66 F46S, Y54H, T93A, T93A S217N,then a ND — 53 ND — ND S167R, S217N, H287A, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 67 TIC807_67 F46S, Y54H, S95A, S95A S217N, then a ND —52 ND — ND S167R, S217N, M300A, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 68 TIC807_68 F46S, Y54H, F147A, F147A S217N, then a ND — 52 ND —ND S167R, S217N, I275A, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 69TIC807_69 F46S, Y54H, S95A, S95A S217N, then a ND — 51 ND — ND S167R,S217N, T247A, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 70 TIC807_70F46S, Y54H, F147A, F147A S217N, then a ND — 50 ND — ND S167R, V175A,S217N, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 71 TIC807_71 F46S, Y54H,F147A, F147A S217N, then a ND — 50 ND — ND S159A, S167R, S217N,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 72 TIC807_72 F46S, Y54H, S95A,S95A, S217N, then a ND — 48 ND — ND F147A, S167R, S217N, F147Acontiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 73 TIC807_73 F46S, Y54H, F147A,F147A S217N, then a ND — 47 ND — ND S167R, L187A, S217N, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 74 TIC807_74 F46S, Y54H, T93A, T93A S217N,then a ND — 47 ND — ND S167R, T182A, S217N, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 75 TIC807_75 F46S, Y54H, F147A, F147A S217N, then a ND —46 ND — ND S167R, S217N, T245A, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 76 TIC807_76 F46S, Y54H, T93A, T93A S217N, then a ND — 46 ND —ND S167R, S217N, S249A, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 77TIC807_77 F46S, Y54H, T93A, T93A, S217N, then a ND — 45 ND — ND Q149A,S167R, S217N, Q149A contiguous triple then a contiguous triple deletionin residue deletion in residue range range 196-201 196-201 78 TIC807_78F46S, Y54H, T93A, T93A, S217N, then a ND — 45 ND — ND S151A, S167R,S217N, S151A contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 79 TIC807_79F46S, Y54H, Q70A, F147A S217N, then a ND — 45 ND — ND F147A, S167R,S217N, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 80 TIC807_80 F46S, Y54H,S95A, S95A S217N, then a ND — 44 ND — ND Q148A, S167R, S217N, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 81 TIC807_81 F46S, Y54H, T93A, T93A S217N,then a ND — 44 ND — ND S167R, S217N, T274A, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 82 TIC807_82 F46S, Y54H, T93A, T93A S217N, then a ND —43 ND — ND S167R, S217N, then a contiguous triple contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 83TIC807_83 F46S, Y54H, T93A, T93A S217N, then a ND — 42 ND — ND S167R,M177A, S217N, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 84 TIC807_84F46S, Y54H, S95A, S95A S217N, then a ND — 42 ND — ND S167R, S217N,V250A, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 85 TIC807_85 F46S, Y54H,T93A, T93A S217N, then a ND — 42 ND — ND E155A, S167R, S217N, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 86 TIC807_86 F46S, Y54H, I134A, F147A S217N,then a ND — 41 ND — ND F147A, S167R, S217N, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 87 TIC807_87 F46S, Y54H, T93A, T93A S217N, then a ND —41 ND — ND S167R, S217N, T245A, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 88 TIC807_88 F46S, Y54H, T93A, T93A S217N, then a ND — 40 ND —ND S167R, S217N, then a contiguous triple contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 followed by afollowed by S198A S198A 89 TIC807_89 F46S, Y54H, F147A, F147A S217N,then a ND — 40 ND — ND S167R, S217N, N295A, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 90 TIC807_90 F46S, Y54H, A87S, F147A S217N, then a ND —38 ND — ND F147A, S167R, S217N, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 91 TIC807_91 F46S, Y54H, F147A, F147A S217N, then a ND — 38 ND —ND S167R, S217N, S249A, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 92TIC807_92 F46S, Y54H, S95A, S95A S217N, then a ND — 37 ND — ND S167R,S217N, I275A, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 93 TIC807_93F46S, Y54H, T93A, T93A S217N, then a ND — 37 ND — ND A139S, S167R,S217N, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 94 TIC807_94 F46S, Y54H,F147A, F147A S217N, then a ND — 36 ND — ND S167R, S217N, P305A,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 95 TIC807_95 F46S, Y54H, F147A,F147A S217N, then a ND — 36 ND — ND S167R, S217N, then a contiguoustriple contiguous triple deletion in residue deletion in residue rangerange 196-201 196-201 followed by a followed by S198A S198A 96 TIC807_96F46S, Y54H, F147A, F147A S217N, then a ND — 36 ND — ND S167R, S217N,S252A, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 97 TIC807_97 F46S, Y54H,T93A, T93A, S217N, then a ND — 36 ND — ND F147A, S167R, S217N, F147Acontiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 98 TIC807_98 F46S, Y54H, F147A,F147A S217N, then a ND — 36 ND — ND S167R, S217N, V250A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 99 TIC807_99 F46S, Y54H, F147A, F147A S217N,then a ND — 35 ND — ND S167R, S217N, T243A, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 100 TIC807_100 F46S, Y54H, F147A, F147A S217N, then a ND— 35 ND — ND S167R, S217N, then a contiguous triple contiguous tripledeletion in residue deletion in residue range range 196-201 196-201followed by a followed Y197A by Y197A ND — 35 ND — ND 101 TIC807_101F46S, Y54H, S95A, S95A S217N, then a S167R, S217N, N295A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 ND — 34 ND — ND 102 TIC807_102 F46S, Y54H,F147A, F147A S217N, then a S167R, S217N, then a contiguous triplecontiguous triple deletion in residue deletion in residue range range196-201 196-201 ND — 32 ND — ND 103 TIC807_103 F46S, Y54H, F147A, F147AS217N, then a S167R, S217N, E299A, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 104 TIC807_104 F46S, Y54H, S95A, S95A S217N, then a ND — 32 ND —ND S167R, S217N, R282A, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 105TIC807_105 F46S, Y54H, T93A, T93A S217N, then a ND — 32 ND — ND S167R,S217N, I306A, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 106 TIC807_106F46S, Y54H, S95A, S95A S217N, then a ND — 30 ND — ND S167R, S217N,S249A, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 107 TIC807_107 F46S,Y54H, A87S, T93A S217N, then a ND — 30 ND — ND T93A, S167R, S217N,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 108 TIC807_108 F46S, Y54H, T93A,T93A S217N, then a ND — 29 ND — ND S159A, S167R, S217N, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 109 TIC807_109 F46S, Y54H, F147A, F147AS217N, then a ND — 28 ND — ND S167R, S217N, T303A, contiguous triplethen a contiguous triple deletion in residue deletion in residue rangerange 196-201 196-201 110 TIC807_110 F46S, Y54H, T93A, T93A S217N, thena ND — 27 ND — ND Q148A, S167R, S217N, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 111 TIC807_111 F46S, Y54H, S95A, S95A S217N, then a ND —27 ND — ND S167R, V175A, S217N, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 112 TIC807_112 F46S, Y54H, F147A, F147A S217N, then a ND — 27 ND— ND S167R, S217N, D241A, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 113TIC807_113 F46S, Y54H, S95A, S95A, S217N, then a ND — 26 ND — ND F147A,S167R, S217N, F147A contiguous triple then a contiguous triple deletionin residue deletion in residue range range 196-201 196-201 114TIC807_114 F46S, Y54H, F147A, F147A S217N, then a ND — 26 ND — ND S167R,M177A, S217N, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 115 TIC807_115F46S, Y54H, F147A, F147A S217N, then a ND — 26 ND — ND S167R, S217N,M300A, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 116 TIC807_116 F46S,Y54H, S95A, S95A S217N, then a ND — 26 ND — ND S167R, S217N, P305A,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 117 TIC807_117 F46S, Y54H, F147A,S217N, then a S167R, S217N, then a F147A contiguous triple ND — 25 ND —ND contiguous triple deletion in residue deletion in residue range range196-201 196-201 followed by followed by H196A H196A 118 TIC807_118 F46S,Y54H, A139S, F147A S217N, then a ND — 25 ND — ND F147A, S167R, S217N,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 119 TIC807_119 F46S, Y54H, T93A,T93A S217N, then a ND — 25 ND — ND S167R, S217N, N295A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 120 TIC807_120 F46S, Y54H, T93A, T93A S217N,then a ND — 24 ND — ND T145A, S167R, S217N, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 121 TIC807_121 F46S, Y54H, S117A, F147A S217N, then a ND— 24 ND — ND F147A, S167R, S217N, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 122 TIC807_122 F46S, Y54H, T93A, T93A S217N, then a ND — 23 ND —ND S167R, S217N, I275A, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 123TIC807_123 F46S, Y54H, S95A, S95A S217N, then a ND — 23 ND — ND S167R,S217N, H287A, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 124 TIC807_124F46S, Y54H, S95A, S95A S217N, then a ND — 21 ND — ND G105A, S167R,S217N, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 125 TIC807_125 F46S,Y54H, S95A, S95A S217N, then a ND — 20 ND — ND S167R, S217N, then acontiguous triple contiguous triple deletion in residue deletion inresidue range range 196-201 196-201 126 TIC807_126 F46S, Y54H, S95A,S95A S217N, then a ND — 20 ND — ND I134A, S167R, S217N, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 127 TIC807_127 F46S, Y54H, T93A, T93A S217N,then a ND — 20 ND — ND S167R, S217N, M300A, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 128 TIC807_128 F46S, Y54H, T93A, T93A S217N, then a ND —18 ND — ND S167R, S217N, T303A, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 129 TIC807_129 F46S, Y54H, T93A, T93A S217N, then a ND — 18 ND —ND A150S, S167R, S217N, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 130TIC807_130 F46S, Y54H, S95A, S95A S217N, then a ND — 18 ND — ND E155A,S167R, S217N, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 131 TIC807_131F46S, Y54H, T93A, T93A S217N, then a ND — 17 ND — ND T145A, S167R,S217N, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 132 TIC807_132 F46S,Y54H, S95A, S95A W208A, S217N, ND — 17 ND — ND S167R, W208A, S217N, thena contiguous then a contiguous triple triple deletion in deletion inresidue range residue range 196-201 196-201 133 TIC807_133 F46S, Y54H,S95A, S95A, S217N, then a ND — 17 ND — ND S167R, T182A, S217N, T182Acontiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 134 TIC807_134 F46S, Y54H, T93A,T93A S217N, then a ND — 17 ND — ND S167R, S217N, T243A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 135 TIC807_135 F46S, Y54H, S95A, S95A S217N,then a ND — 16 ND — ND S167R, S217N, I306A, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 136 TIC807_136 F46S, Y54H, S95A, S95A S217N, then a ND —16 ND — ND S117A, S167R, S217N, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 137 TIC807_137 F46S, Y54H, T93A, T93A S217N, then a ND — 15 ND —ND S119A, S167R, S217N, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 138TIC807_138 F46S, Y54H, T68A, F147A S217N, then a ND — 15 ND — ND F147A,S167R, S217N, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 ND 139TIC807_139 F46S, Y54H, G105A, F147A S217N, then a ND — 15 ND — F147A,S167R, S217N, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 140 TIC807_140F46S, Y54H, S95A, S95A S217N, then a ND — 15 ND — ND E125A, S167R,S217N, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 141 TIC807_141 F46S,Y54H, T93A, T93A S217N, then a ND — 14 ND — ND E155A, S167R, S217N,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 142 TIC807_142 F46S, Y54H, T93A,T93A S217N, then a ND — 14 ND — ND S167R, S217N, P305A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 143 TIC807_143 F46S, Y54H, S95A, S95A S217N,then a ND — 13 ND — ND S167R, M177A, S217N, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 144 TIC807_144 F46S, Y54H, T93A, T93A S217N, then a ND —13 ND — ND S167R, S217N, then a contiguous triple contiguous tripledeletion in residue deletion in residue range range 196-201 196-201followed by followed by H196A H196A 145 TIC807_145 F46S, Y54H, T93A,T93A S217N, then a ND — 11 ND — ND D153A, S167R, S217N, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201-T93A-D153A 146 TIC807_146 F46S, Y54H, F147A,F147A W208A, S217N, ND — 10 ND — ND S167R, S217N, W208A, then acontiguous then a contiguous triple triple deletion in deletion inresidue range residue range 196-201 196-201 147 TIC807_147 F46S, Y54H,T93A, T93A S217N, then a ND —  9 ND — ND S167R, S217N, I306A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 148 TIC807_148 F46S, Y54H, S95A, S95A S217N,then a ND —  9 ND — ND S167R, S217N, then a contiguous triple contiguoustriple deletion in residue deletion in residue range range 196-201196-201 followed by followed by H196A H196A 149 TIC807_149 F46S, Y54H,S95A, S95A, S217N, then a ND —  9 ND — ND Q149A, S167R, S217N, Q149Acontiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 150 TIC807_150 F46S, Y54H, S95A,S95A S217N, then a ND —  8 ND — ND S167R, S217N, S293A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 151 TIC807_151 F46S, Y54H, S95A, S95A S217N,then a ND —  8 ND — ND A150S, S167R, S217N, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 152 TIC807_152 F46S, Y54H, S119A, F147A S217N, then a ND—  7 ND — ND F147A, S167R, S217N, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 153 TIC807_153 F46S, Y54H, S95A, S95A S217N, then a ND —  7 ND —ND S167A, S217N, then a contiguous triple contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 154 TIC807_154F46S, Y54H, F147A, F147A S217N, then a ND —  7 ND — ND I158A, S167R,S217N, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 155 TIC807_155 F46S,Y54H, S95A, S95A S217N, then a ND —  7 ND — ND S167R, S217N, M300A,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 156 TIC807_156 F46S, Y54H, F147A,F147A, S217N, then a ND —  6 ND — ND T182A, S167R, S217N, T182Acontiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 157 TIC807_157 F46S, Y54H, F147A,F147A S217N, then a ND —  5 ND — ND S167R, S217N, F235A, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 158 TIC807_158 F46S, Y54H, F147A, F147AS217N, then a ND —  5 ND — ND S167A, S217N, then a contiguous triplecontiguous triple deletion in residue deletion in residue range range196-201 196-201 159 TIC807_159 F46S, Y54H, T93A, T93A S217N, then a ND — 5 ND — ND S167R, S217N, D241A, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 160 TIC807_160 F46S, Y54H, S95A, S95A S217N, then a ND —  4 ND —ND S167R, S217N, T274A, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 161TIC807_161 F46S, Y54H, T93A, T93A S217N, then a ND —  4 ND — ND Q148A,S167R, S217N, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 162 TIC807_162F46S, Y54H, S95A, S95A S217N, then a ND —  4 ND — ND S167R, S217N,D241A, contiguous triple then a contiguous triple deletion in residuedeletion in residue range range 196-201 196-201 163 TIC807_163 F46S,Y54H, S95A, S95A S217N, then a ND —  4 ND — ND E155A, S167R, S217N,contiguous triple then a contiguous triple deletion in residue deletionin residue range range 196-201 196-201 164 TIC807_164 F46S, Y54H, T93A,T93A S217N, then a ND —  4 ND — ND S167R, L187A, S217N, contiguoustriple then a contiguous triple deletion in residue deletion in residuerange range 196-201 196-201 165 TIC807_165 F46S, Y54H, T93A, T93A S217N,then a ND —  4 ND — ND S167R, S217N, T303A, contiguous triple then acontiguous triple deletion in residue deletion in residue range range196-201 196-201 166 TIC807_166 F46S, Y54H, T93A, T93A S217N, then a ND — 3 ND — ND E155A, S167R, S217N, contiguous triple then a contiguoustriple deletion in residue deletion in residue range range 196-201196-201 167 TIC807_167 F46S, Y54H, T93A, T93A S217N, then a ND —  3 ND —ND D153A, S167R, S217N, contiguous triple then a contiguous tripledeletion in residue deletion in residue range range 196-201 196-201 168TIC807_168 F46S, Y54H, T93A, T93A S217N, then a ND —  3 ND — ND I134A,S167R, S217N, contiguous triple then a contiguous triple deletion inresidue deletion in residue range range 196-201 196-201 169 TIC807_169N137T none None ND —  44* ND — ND 169 TIC807_169 E135S none None ND — 20* ND — ND 169 TIC807_169 N137T, E135S none None ND —  34* ND — ND 169TIC807_169 E135T, N137D none None ND —  16* ND — ND 169 TIC807_169 E133Enone None ND —  60* ND — ND 169 TIC807_169 E135A, N137G none None ND — 50* ND — ND 170 TIC807_170 E125C none None ND — 12 ND — ND 170TIC807_170 E125H none None ND — 38 ND — ND 170 TIC807_170 E125R noneNone ND — 14 ND — ND 170 TIC807_170 E125F none None ND — 33 ND — ND 170TIC807_170 E125S none None ND — 24 ND — ND 170 TIC807_170 E125Q noneNone ND — 21 ND — ND 170 TIC807_170 E125K none None ND — 20 ND — ND 170TIC807_170 E125T none None ND — 33 ND — ND 170 TIC807_170 E125N noneNone ND — 19 ND — ND 170 TIC807_170 E125A none None ND — 41 ND — ND 170TIC807_170 E125L none None ND — 13 ND — ND 170 TIC807_170 E125V noneNone ND — 14 ND — ND 170 TIC807_170 E125M none None ND — 13 ND — ND 170TIC807_170 E125D none None ND — 15 ND — ND 170 TIC807_170 E125Y noneNone ND — 38 ND — ND 171 TIC807_171 T133E none None ND — 23 ND — ND 171TIC807_171 T133Y none None ND — 17 ND — ND 171 TIC807_171 T133W noneNone ND — 13 ND — ND 172 TIC807_172 I134V none None ND — 18 ND — ND 172TIC807_172 I134L none None ND — 10 ND — ND 172 TIC807_172 I134F noneNone ND — 18 ND — ND 172 TIC807_172 I134K none None ND — 13 ND — ND 172TIC807_172 I134C none None ND — 30 ND — ND 172 TIC807_172 I134M noneNone ND — 33 ND — ND 173 TIC807_173 E135V none None ND — 13 ND — ND 173TIC807_173 E135W none None ND — 13 ND — ND 173 TIC807_173 E135T noneNone ND — 39 ND — ND 174 TIC807_174 N137H none None ND — 42 ND — ND 174TIC807_174 N137Y none None ND — 17 ND — ND 174 TIC807_174 N137T noneNone ND — 31 ND — ND 174 TIC807_174 N137E none None ND — 32 ND — ND 174TIC807_174 N137S none None ND — 24 ND — ND 174 TIC807_174 N137A noneNone ND — 24 ND — ND 174 TIC807_174 N137Q none None ND — 21 ND — ND 174TIC807_174 N137G none None ND — 18 ND — ND 174 TIC807_174 N137I noneNone ND — 10 ND — ND 174 TIC807_174 N137W none None ND — 17 ND — ND 174TIC807_174 N137K none None ND — 66 ND — ND 174 TIC807_174 N137C noneNone ND — 19 ND — ND 174 TIC807_174 N137M none None ND — 41 ND — ND 174TIC807_174 N137D none None ND — 69 ND — ND 174 TIC807_174 N137F noneNone ND — 13 ND — ND 174 TIC807_174 N137R none None ND — 37 ND — ND 175TIC807_175 F147V F147V None ND — 87 ND — ND 175 TIC807_175 F147T F147TNone ND — 68 ND — ND 175 TIC807_175 F147C F147C None ND — 74 ND — ND 175TIC807_175 F147L F147L None ND — 62 ND — ND 175 TIC807_175 F147D F147DNone ND — 51 ND — ND 175 TIC807_175 F147A F147A None ND — 57 ND — ND 175TIC807_175 F147G F147G None ND — 56 ND — ND 175 TIC807_175 F147E F147ENone ND — 50 ND — ND 175 TIC807_175 F147I F147I None ND — 69 ND — ND 175TIC807_175 F147Y F147Y None ND — 67 ND — ND 175 TIC807_175 F147M F147MNone ND — 64 ND — ND 175 TIC807_175 F147N F147N None ND — 64 ND — ND 175TIC807_175 F147Q F147Q None ND — 50 ND — ND 175 TIC807_175 F147H F147HNone ND — 60 ND — ND 175 TIC807_175 F147R F147R None ND — 20 ND — ND 175TIC807_175 F147W F147W None ND — 82 ND — ND 175 TIC807_175 F147P F147PNone ND —  7 ND — ND 176 TIC807_176 Q149D Q149D None ND — 92 ND — ND 176TIC807_176 Q149E Q149E None ND — 89 ND — ND 176 TIC807_176 Q149C Q149CNone ND — 87 ND — ND 176 TIC807_176 Q149A Q149A None ND — 76 ND — ND 176TIC807_176 Q149F Q149F None ND — 54 ND — ND 177 TIC807_177 A150S noneNone ND — 34 ND — ND 177 TIC807_177 A150L none None ND — 24 ND — ND 177TIC807_177 A150V none None ND — 25 ND — ND 177 TIC807_177 A150G noneNone ND — 28 ND — ND 177 TIC807_177 A150D none None ND — 19 ND — ND 177TIC807_177 A150W none None ND — 13 ND — ND 177 TIC807_177 A150E noneNone ND — 24 ND — ND 177 TIC807_177 A150N none None ND — 18 ND — ND 177TIC807_177 A150Y none None ND — 11 ND — ND 177 TIC807_177 A150F noneNone ND — 11 ND — ND 177 TIC807_177 A150P none None ND — 11 ND — ND 177TIC807_177 A150K none None ND — 17 ND — ND 177 TIC807_177 A150T noneNone ND — 17 ND — ND 177 TIC807_177 A150Q none None ND — 11 ND — ND 177TIC807_177 A150R none None ND — 11 ND — ND 178 TIC807_178 E155C noneNone ND — 82 ND — ND 178 TIC807_178 E155I none None ND — 36 ND — ND 178TIC807_178 E155K none None ND — 28 ND — ND 178 TIC807_178 E155D noneNone ND — 22 ND — ND 178 TIC807_178 E155H none None ND — 22 ND — ND 178TIC807_178 E155Y none None ND — 16 ND — ND 178 TIC807_178 E155Q noneNone ND — 16 ND — ND 178 TIC807_178 E155L none None ND — 15 ND — ND 178TIC807_178 E155N none None ND — 14 ND — ND 178 TIC807_178 E155T noneNone ND — 13 ND — ND 178 TIC807_178 E155A none None ND — 11 ND — ND 178TIC807_178 E155F none None ND —  7 ND — ND 178 TIC807_178 E155R noneNone ND —  6 ND — ND 178 TIC807_178 E155M none None ND —  6 ND — ND 178TIC807_178 E155P none None ND —  5 ND — ND 178 TIC807_178 E155W noneNone ND —  5 ND — ND 178 TIC807_178 E155S none None ND —  4 ND — ND 178TIC807_178 E155V none None ND —  4 ND — ND 179 TIC807_179 N157C noneNone ND — 86 ND — ND 179 TIC807_179 N157D none None ND — 64 ND — ND 179TIC807_179 N157W none None ND — 52 ND — ND 179 TIC807_179 N157Y noneNone ND — 39 ND — ND 179 TIC807_179 N157M none None ND — 22 ND — ND 179TIC807_179 N157A none None ND — 22 ND — ND 179 TIC807_179 N157F noneNone ND — 20 ND — ND 179 TIC807_179 N157V none None ND — 18 ND — ND 179TIC807_179 N157L none None ND — 18 ND — ND 179 TIC807_179 N157P noneNone ND — 18 ND — ND 179 TIC807_179 N157E none None ND — 17 ND — ND 179TIC807_179 N157T none None ND —  8 ND — ND 179 TIC807_179 N157G noneNone ND —  7 ND — ND 179 TIC807_179 N157I none None ND —  7 ND — ND 179TIC807_179 N157R none None ND —  6 ND — ND 202 TIC807_M8_ F46S, Y54H,S95A, S95A, S217N, then a ND —  92* ND — ND d5C F147A, S167R, S217N,F147A contiguous triple then a five amino acid deletion in residuedeletion at 305-309, range 196-201 then a contiguous triple deletion inresidue range 196-201 204 TIC807_M14 F46S, Y54H, S95A, S95A, P219R, thena ND — — ND —  60* F147S, Q149E, S167R, F147A, contiguous triple P219R,S249R, V251E, Q149E, deletion in residue Q253R, R273W, then a V251E,range 196-201 contiguous triple Q253R deletion in residue range 196-201ND = Not Determined; *tested at about 5 μg/mL.

Example 5: Insect Inhibitory Activities of Protein Members of thePresent Invention

Proteins of the present invention, such as but not limited to TIC807 M1(SEQ ID NO:6), TIC807_M2 (SEQ ID NO:8), TIC807_M3 (SEQ ID NO:10),TIC807_M4 (SEQ ID NO:12), TIC807_M5 (SEQ ID NO:14), TIC807_M6 (SEQ IDNO:16), TIC807_M7 (SEQ ID NO:18), TIC807_M8 (SEQ ID NO:20), TIC807_M9(SEQ ID NO:22), TIC807_M14 (SEQ ID NO:32), TIC807_M15 (SEQ ID NO:34),and TIC807_M16 (SEQ ID NO:36), are prepared and tested for bioactivityagainst pests of plants other than from Lygus.

Proteins TIC807_M10 (SEQ ID NO:24), TIC807_M11 (SEQ ID NO:26),TIC807_M12 (SEQ ID NO:28), and TIC807_M13 (SEQ ID NO:30) were preparedand tested for bioactivity against pests from the order Lepidoptera,Coleoptera, Heteroptera, and Homoptera. Protein TIC807_M5 (SEQ ID NO:14)was prepared and tested for bioactivity against Coleopteran pests.Bioassays were conducted to evaluate the effects of these proteins oninsects as shown in Table 5. Feeding assays were conducted on anartificial diet containing the insecticidal protein. The insecticidalprotein was prepared as described in example 3 and topically appliedusing an insect-specific artificial diet, depending on the insect beingtested. The toxin was suspended in a buffer and applied at a rate of 500μg/mL of sample per well, and in the case of TIC807_M5 of 1000 μg/mL,and then allowed to dry. Mean stunting scores and population mortalitieswere determined on three populations of 8 insects per insect speciestested. Results were expressed as positive (+) for insect reactions suchas stunting and mortality that were statistically significant comparedto the untreated control. Results were expressed as negative (−) if theinsects were similar to the UTC, that is, feeding diet to which theabove buffer only has been applied.

TABLE 5 eHTP's demonstrate additional insect inhibitory activitiesagainst pests other than Lygus spp. Protein μg/mL CPB WCR ECB SWCB CEWFAW SGSB NBSB GPA UTC 0 − − − − − − − − − TIC807_M5 1000 + − ND ND ND NDND ND ND TIC807_M10 500 − − − − − − − − − TIC807_M11 500 + − − − − − − −− TIC807_M12 500 − − − − − − − − − TIC807_M13 500 + − − − − − − − − UTC= UnTreated Control; ND = Not Determined CPB = Colorado potato beetle(Leptinotarsa decemlineata); WCR = western corn rootworm (Diabroticavirgifera); ECB = European corn borer (Ostrinia nubilalis); southwesterncorn borer (Diatraea grandiosella); CEW = corn earworm (Helicoverpazea); FAW = Fall armyworm (Spodoptera frugiperda); SGSB = southern greenstink bug (Nezara virudula); NBSB = neotropical brown stink bug(Euschistus heros); GPA = Green peach aphid (Myzus persicae).

The proteins of the present invention are also tested for bioactivityagainst a pest from the phylum Nematoda.

Example 6: Plants Expressing Proteins of the Present Invention ExhibitInsect Inhibitory Activity

This example illustrates expression of proteins of the present inventionin plants, and demonstrates that cotton plants expressing proteins ofthe present invention exhibit insect inhibitory activity.

Polynucleotide segments for use in expression of the proteins of thepresent invention in plants are made according to the methods set forthin U.S. Pat. No. 7,741,118. For example, toxin proteins having the aminoacid sequence as set forth in SEQ ID NO:4 (TIC807_4), SEQ ID NO:6(TIC807_M1), SEQ ID NO:8 (TIC807_M2), SEQ ID NO:10 (TIC807_M3), SEQ IDNO:12 (TIC807_M4), SEQ ID NO:14 (TIC807_M5), SEQ ID NO:16 (TIC807_M8),SEQ ID NO:18 (TIC807_M6), SEQ ID NO:20 (TIC807_M7), SEQ ID NO:22 (TIC80722), SEQ ID NO:24 (TIC807_24), SEQ ID NO:26 (TIC807_26), SEQ ID NO:28(TIC807_M9), SEQ ID NO:30 (TIC807_M10), SEQ ID NO:32 (TIC807_M11), andSEQ ID NO:34 (TIC807_M13), are expressed from polynucleotide segmentsdesigned for use in plants and encoding the proteins of the presentinvention, including the polynucleotide sequences as set forth in SEQ IDNO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195,SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ IDNO:200, and SEQ ID NO:201, respectively.

It is intended that polynucleotide segments (or polynucleotidemolecules) encoding each of the variant proteins or insect inhibitoryfragments thereof, be used alone or in combination with each other, orin combination with other insect inhibitory proteins or insectinhibitory agents such as dsRNA mediated gene suppression molecules.Such combinations designed to work in synergistic or compatiblemechanism with the proteins of the present invention. The intention ofthese combinations is to achieve plants and plant cells protected frompest, particularly insect pest, infestation. The specific variantproteins within the scope of the invention include the proteinscorresponding to SEQ ID NOs listed in Table 4B and described throughoutthe application as filed.

Polynucleotide segments from SEQ ID NO:188 (encodes for TIC807_M2, SEQID NO:8) and from SEQ ID NO:192 (encodes for TIC807_M8, SEQ ID NO:16)were each recombinantly engineered into expression constructs for cottontransformation.

Transgenic cotton plants (recombinant cotton plants) were produced andtested for efficacy. Regenerated (R0) transgenic plants were selectedthat were low in copy number and high in expression of the respectivevariant protein, as determined by various quantitative andsemi-quantitative methods, e.g. PCR, ELISAs and Westerns. Expressionlevels in R0 cotton leaf tissue typically ranged from 0.5 to 500 ppmfresh weight. R0 plants expressing high levels of protein weretransferred to soil and selfed. Thirty seed from each of the selfed R0plants were planted and progeny homozygous for the transgene were grownto flowering. Eleven to 18 plants per 4 to 5 events per each constructof this example were tested for efficacy against Lygus (Tables 6A, 6B,and 6C). The untransformed cotton cultivar, plants from the poolednegative segregate population (progeny not containing the transgene),and plants expressing TIC807 parent protein served as negative controls.A branch of a flowering stage cotton plant was enclosed in a mesh bagmade from breathable plastic ‘pollination’ sleeves (Vilutis and Co.Inc., Frankfort, Ill.), and multiple branches set up in similar fashion.Each mesh bag was secured at the stem using a twist tie. About 4-6 Lygushesperus nymphs (<24 hours post-hatch) were placed into a 1.4 ml conicaltube (Matrix Technologies Corp., NH). The branch inside a mesh bag wasinfested with nymphs by sliding the uncapped conical tube into the meshbag. Insects were allowed to feed for a period of 10-11 days before allsurviving insects in the mesh bag were collected on dry ice. Survivorswere weighed to obtain a gross mass. Percent mortality and mean survivormass were calculated. Missing insects were included in the mortalitypercent mortality calculation. As shown in Tables 6A, 6B, and 6C, cottonplants expressing the variant proteins TIC807_M2 and TIC807_M8significantly impacted the growth and development of Lygus hesperusnymphs. Based on these results, these plants, seed, expressionconstructs were advanced for further development.

TABLE 6A Mean % mortality determined from flowering stage Lygus feedingassays with cotton plants expressing the variant TIC807 proteinsTIC807_M2 and TIC807_M8. Plant Event Mean % Mortality ID Protein NMortality Std Dev SEM Lo 95% Up 95% t group 64 TIC807_M8 18 78.88919.967 4.706 68.959 88.818 A 49 TIC807_M8 18 75.556 22.288 5.253 64.47286.639 A 91 TIC807_M2 18 74.444 20.356 4.798 64.321 84.567 A 20TIC807_M8 18 73.333 19.403 4.573 63.685 82.982 A 15 TIC807_M8 18 66.66725.668 6.050 53.902 79.431 AB 58 TIC807_M2 18 65.556 19.166 4.517 56.02575.086 AB 48 TIC807_M2 18 64.444 21.206 4.998 53.899 74.990 AB 19TIC807_M2 18 53.333 25.668 6.050 40.569 66.098 BC 68 TIC807_M2 18 47.77825.795 6.080 34.950 60.605 C Negative 24 41.667 22.001 4.491 32.37650.957 C

TABLE 6B Mean Instar determined from flowering stage Lygus feedingassays with cotton plants expressing the variant TIC807 proteinsTIC807_M2 and TIC807_M8. Mean Instar Plant Event ID Construct N InstarStd Dev SEM Lo 95% Up 95% t group 64 TIC807_M8 11 3.636 0.552 0.1663.266 4.007 C 68 TIC807_M2 16 3.949 0.803 0.201 3.521 4.377 BC 48TIC807_M2 16 4.042 0.604 0.151 3.720 4.364 BC 58 TIC807_M2 17 4.0690.802 0.194 3.657 4.481 BC 15 TIC807_M8 15 4.094 0.747 0.193 3.681 4.508BC 19 TIC807_M2 17 4.100 0.698 0.169 3.741 4.459 BC 91 TIC807_M2 124.125 0.829 0.239 3.598 4.652 ABC 49 TIC807_M8 12 4.139 0.762 0.2203.655 4.623 ABC 20 TIC807_M8 14 4.298 0.918 0.245 3.768 4.828 ABNegative 24 4.599 0.774 0.158 4.273 4.926 A

TABLE 6C Mean Survival Mass determined from flowering stage Lygusfeeding assays with cotton plants expressing the variant TIC807 proteinsTIC807_M2 and TIC807_M8. Mean Survivor Survival Mass Plant Event IDConstruct N Mass Std Dev SEM Lo 95% Up 95% t group 64 TIC807_M8 11 2.3151.489 0.449 1.314 3.315 C 68 TIC807_M2 16 3.548 1.325 0.331 2.843 4.254B 58 TIC807_M2 17 3.561 1.348 0.327 2.868 4.255 B 48 TIC807_M2 16 3.5961.436 0.359 2.831 4.362 B 91 TIC807_M2 12 3.775 1.775 0.512 2.647 4.902AB 49 TIC807_M8 12 3.837 2.135 0.616 2.481 5.193 AB 19 TIC807_M2 173.908 1.467 0.356 3.154 4.662 AB 20 TIC807_M8 14 3.918 1.950 0.521 2.7925.044 AB 15 TIC807_M8 15 3.937 1.906 0.492 2.881 4.993 AB Negative 244.735 1.179 0.241 4.237 5.233 A Std Dev—standard deviation SEM—Standarderror on the mean Lo 95% = Lower limit at 95% confidence interval Up 95%= Upper limit at 95% confidence interval T grouping = Using a leastsignificant difference test, F value = 101.1756, df = 15, 44, Pr <0.0001

In another example, cotton plants from five transgenic events expressingTIC807_M11 were tested in a field trial having natural Lygus infestationpressures. These plants demonstrated field efficacy compared to thenon-transgenic recipient line (DP393 germplasm used for transformation).The average number of Lygus lineolaris insects on five plants per eventwas significantly lower than the average number of Lygus lineolarisinsects on plants from the non-transgenic control. Seed cotton yieldfrom plants from the five events was statistically comparable to seedcotton yield of the non-transgenic control, e.g. season-long squareretention.

In another similar field trial, cotton plants from seven transgenicevents expressing TIC807_M10 demonstrated field efficacy compared to thenon-transgenic control. The average number of Lygus lineolaris insectson five plants per event was significantly lower than the average numberof Lygus lineolaris insects on plants from the non-transgenic control.Seed cotton yield from plants from three of the seven events wasstatistically higher than to seed cotton yield of the non-transgeniccontrol.

In another example, cotton plants from thirty-four transgenic eventsexpressing TIC807_M13 demonstrated growth chamber efficacy compared tothe non-transgenic control. Mesh bags were placed around the wholecotton plants at flowering stage (instead of just around single branchesdescribed earlier in this example). Five plants per event were evaluatedand the average number of Lygus lineolaris insects recovered (nymphs toadults to 2^(nd) generation Lygus) per plant was significantly lowerthan the average number of Lygus lineolaris insects per non-transgenicplant.

Similar experiments are performed with plants expressing proteins listedin Table 1 and in Tables 4A and 4B.

Example 7: Tissue from Alfalfa Plants Expressing Proteins of the PresentInvention Exhibit Insect Inhibitory Activity

This example illustrates expression of proteins of the present inventionin alfalfa plants, and demonstrates that tissue from alfalfa plantsexpressing proteins of the present invention exhibit insect inhibitoryactivity.

Polynucleotide segment from SEQ ID NO:192 (encodes for TIC807_M8, SEQ IDNO:16) was recombinantly engineered into three differently configuredexpression constructs for alfalfa transformation. For purposes of datareporting, the three recombinant constructs are coded [ER], [ES], and[ET].

Transgenic alfalfa plants (recombinant alfalfa plants) were recoveredfrom transformants that were outcrossed and then selfed. Recombinantalfalfa plants were selected that were low in copy number and high inTIC807 expression as determined by RT-PCR and semi-quantitative Westernmethods, respectively. Alfalfa plant tissue from ten separate eventswere pooled, lyophilized, ground, and resuspended in stock buffer, 25 mMNaCarb, pH 10.5. Plant tissue from Alfalfa having no TIC807_M8expressing transgene was prepared for use as control. Stock preparationswere serially diluted 100, 300, and 900 fold for incorporation intoLygus diet. Using the feeding assay method of Example 4, mortality andstunting scores were determined on day 5 and compared to controls (SeeTables 7A and 7B; data were analyzed using JMP4 statistical software).For each test sample and each dilution, three populations of eightnymphs were subjected to this bioassay. Stunting scores correspond tovisual mass ratings where 0=no difference to negative control, 1=about25% less mass, 2=about 50% less mass, and 3=about 75% less mass. Theaverage of the stunting scores for each population of eight nymphs isreported.

TABLE 7A Mean % mortality determined from Lygus feeding assays with dietincorporated with tissue from alfalfa plants expressing the variantTIC807 protein TIC807_M8. Mean Dilution Stunting Lower Upper t ConstructSample Source fold Score Std Dev SEM 95% 95% Grouping [ER] PooledAlfalfa 100 2.00 0.00 0.00 2.00 2.00 CD TIC807_M8 tissue from 10 3000.00 0.00 0.00 0.00 0.00 E events per construct 900 0.00 0.00 0.00 0.000.00 E [ES] 100 3.00 0.00 0.00 3.00 3.00 A TIC807_M8 300 2.33 0.58 0.330.90 3.77 BC 900 0.00 0.00 0.00 0.00 0.00 E [ET] 100 2.67 0.58 0.33 1.234.10 AB TIC807_M8 300 1.67 0.58 0.33 0.23 3.10 D 900 0.00 0.00 0.00 0.000.00 E None Control Alfalfa 100 2.00 0.00 0.00 2.00 2.00 CD 300 0.000.00 0.00 0.00 0.00 E 900 0.00 0.00 0.00 0.00 0.00 E No Alfalfaincorporated in the diet 0 0.00 0.00 0.00 0.00 0.00 E

TABLE 7B Mean stunting determined from Lygus feeding assays with dietincorporated with tissue from alfalfa plants expressing the variantTIC807 protein TIC807_M8. Mean Dilution Percent Lower Upper t ConstructSample Source fold mortality Std Dev SEM 95% 95% Grouping [ER] PooledAlfalfa 100 4.17 7.22 4.17 −13.76 22.09 CD TIC807_M8 tissue from 10 3000.00 0.00 0.00 0.00 0.00 CD events per construct 900 13.10 12.54 7.24−18.06 44.25 CD [ES] 100 56.55 6.27 3.62 40.97 72.12 AB TI807_M8 30041.67 19.09 11.02 −5.77 89.10 B 900 0.00 0.00 0.00 0.00 0.00 CD [ET] 10064.88 19.91 11.50 15.42 114.34 A TIC807_M8 300 16.67 19.09 11.02 −30.7764.10 C 900 12.50 12.50 7.22 −18.55 43.55 CD None Control Alfalfa 10012.50 12.50 7.22 −18.55 43.55 CD 300 0.00 0.00 0.00 0.00 0.00 CD 9008.33 14.43 8.33 −27.52 44.19 CD No Alfalfa incorporated in the diet 02.50 7.01 1.81 −1.38 6.38 D

Example 8: Plants Co-Expressing an eHTP and a Second Insect InhibitoryProtein Exhibiting Lygus Species Inhibitory Activity

Protein samples were prepared containing various mixtures of TIC1415 andTIC807_M13 and tested in bioassay. The TIC1415 protein and other Lygusinhibitory proteins are described in PCT Patent Application PublicationNo. WO 2012/139004. Sample mixtures were fed to Lygus lineolaris usingbioactivity assay. TIC1415 protein alone and TIC807_M13 alone were alsoprepared as positive controls. Buffer was used as negative control.Samples from all three types of preparations exhibited mortality againstLygus lineolaris and survivors were stunted. Mortality and stuntingscores were significant compared to bioactivity scores of insects fedwith buffer (see Table 8A). The data suggests that there are noantagonistic effects. Additional bioassay tests are performed onmixtures to demonstrate synergistic and/or additive effects.

TABLE 8A Bioassay data for protein mix: TIC1415 combined with TIC807_M13Mean† Mean† TIC1415 TIC807_M13 Population T Grouping stunting‡ TGrouping SAMPLE (μg/mL) (μg/mL) mortality on mort score on stuntingTIC1415 + 4.35 1 21.79 AB* 0.60 AB* TIC807_M13 TIC1415 + 2.175 1 20.36B* 0.60 AB* TIC807_M13 TIC1415 + 1.0875 1 12.50 BC 0.60 AB* TIC807_M13TIC1415 + 4.35 0.5 32.50 A* 0.80 A* TIC807_M13 TIC1415 + 1.75 0.265 7.86CD 0.40 ABC TIC807_M13 TIC1415 + 0.875 0.265 0.00 D 0.00 C TIC807_M13TIC1415 + 0.4375 0.265 5.36 CD 0.00 C TIC807_M13 TIC1415 + 4.35 0.2513.21 BC 0.40 ABC TIC807_M13 TIC1415 + 1.75 0.1325 0.00 D 0.00 CTIC807_M13 TIC1415 + 1.75 0.06625 0.00 D 0.00 C TIC807_M13 TIC1415 4.350 12.50 BC 0.40 ABC TIC1415 1.75 0 7.86 CD 0.00 C TIC807_M13 0 1 0.00 D0.20 BC TIC807_M13 0 0.265 2.50 CD 0.00 C Buffer (negative) 0 0 0.00 D0.00 C control †Average (mean) of 5 populations of 8 nymphs perpopulation. ‡Stunting scores correspond to visual mass ratings where 0 =no difference to negative control, 1 = about 25% less mass, 2 = about50% less mass, and 3 = about 75% less mass. The average of the stuntingscores for each population of eight nymphs is reported. *At 95%confidence interval.

Cotton plants comprising events with transgenic DNA were designed toco-express respective proteins TIC1415 and TIC807_M13. Such plants wereevaluated in a caged whole plant assay infested with Lygus lineolaris.Five plants each from ten events were caged and infested with 2 pairs ofmale and female L. lineolaris per plant. The assay was incubated in agrowth chamber under normal environmental conditions for cotton plantdevelopment for 21 days. DP393 negative control plants were grown insimilar manner. At the end of the 3 week period, Lygus of various stagesof development were counted. The mean number per plant of Lygus hesperusinsects at each stage in development were calculated (see Table 8B).

TABLE 8B In-planta data for for protein mix: TIC1415 combined withTIC807_M13 Mean Mean Mean Mean Mean Live Total 3rd 4th 5th 2nd 2ndInstar Instar Instar Gen. Gen. Tukey Construct Event N or < NymphsNymphs Adults Lygus SEM Grouping 12 021 5 0.00 0.00 0.00 0.00 0.00 0.00B 625 5 0.20 0.20 0.20 0.00 0.60 0.24 B 830 5 2.20 0.20 0.00 0.00 2.401.12 AB 890 5 4.40 0.00 0.20 0.00 4.60 2.62 AB 521 5 4.60 0.60 0.00 0.005.20 4.27 AB 980 5 3.40 1.20 1.20 0.00 5.80 4.86 AB 13 426 5 0.00 0.000.00 0.00 0.00 0.00 B 611 5 0.60 0.00 0.00 0.00 0.60 0.60 B 999 5 0.400.00 0.40 0.00 0.80 0.37 B 356 5 6.20 0.00 0.40 0.00 6.60 4.73 AB InbredDP393 10 7.00 2.50 0.80 0.00 10.30 3.75 A (Negative)

What is claimed is:
 1. An insect inhibitory recombinant polypeptidecomprising the amino acid sequence as set forth in SEQ ID NO:
 36. 2. Theinsect inhibitory recombinant polypeptide of claim 1, wherein thepolypeptide exhibits inhibitory activity against an insect species ofthe order Hemiptera.
 3. The insect inhibitory recombinant polypeptide ofclaim 2, wherein the Hemipteran species is selected from the groupconsisting of a Lygus sp., an Emrasca sp., and an Amrasca sp.
 4. Theinsect inhibitory recombinant polypeptide of claim 2, wherein theHemipteran species is selected from the group consisting of LygusHesperus, Lygus lineolaris, and Amrasca devastans.
 5. A polynucleotideencoding a pesticidal polypeptide or pesticidal fragment thereof,wherein the pesticidal polypeptide comprises the amino acid sequence asset forth in SEQ ID NO:
 36. 6. The polynucleotide of claim 5, whereinthe polynucleotide comprises the nucleotide sequence as set forth in SEQID NO:
 35. 7. A host cell comprising the polynucleotide of claim 5,wherein the host cell is selected from the group consisting of abacterial host cell and a plant host cell.
 8. The host cell of claim 7,wherein the bacterial host cell is selected from the group consisting ofAgrobacterium, Rhizobium, Bacillus, Escherichia, Pseudomonas, andSalmonella; and wherein the Bacillus species is a Bacillusthuringiensis, and the Escherichia is an Escherichia coli.
 9. The hostcell of claim 7, wherein the plant host cell is selected from the groupconsisting of a monocot cell and a dicot cell.
 10. An insect inhibitorycomposition comprising the insect inhibitory recombinant polypeptide ofclaim
 1. 11. The composition of claim 10, wherein the composition isprepared by lyophilization, extraction, filtration, or centrifugation.12. The insect inhibitory composition of claim 10, further comprising atleast one insect inhibitory agent different from the insect inhibitoryrecombinant polypeptide.
 13. The insect inhibitory composition of claim12, wherein the at least one insect inhibitory agent is selected fromthe group consisting of an insect inhibitory protein, an insectinhibitory dsRNA molecule, and an insect inhibitory chemistry.
 14. Theinsect inhibitory composition of claim 12, wherein the at least onepesticidal agent exhibits activity against one or more pest species ofthe order Lepidoptera, Coleoptera, Hemiptera, or Homoptera.
 15. A seedcomprising the polynucleotide of claim
 5. 16. The seed of claim 15,wherein the polynucleotide comprises the sequence as set forth in SEQ IDNO:
 35. 17. A method of controlling a Hemipteran pest, the methodcomprising presenting the Hemipteran pest with an inhibitory amount ofthe insect inhibitory recombinant polypeptide of claim
 1. 18. The methodof claim 17, wherein said presenting is via expressing the insectinhibitory recombinant polypeptide in a cotton plant.
 19. A transgenicplant cell, plant, or plant part comprising the insect inhibitoryrecombinant polypeptide of claim
 1. 20. A method of controlling aHemipteran pest, comprising exposing the Hemipteran pest to thetransgenic plant cell, plant or plant part of claim 19, wherein theplant cell, plant or plant part expresses a Hemipteran inhibitory amountof the insect inhibitory recombinant polypeptide.
 21. A commodityproduct obtained from the plant cell, plant, or plant part of claim 19,wherein the commodity product comprises a detectable amount of theinsect inhibitory recombinant polypeptide, wherein the commodity productis selected from the group consisting of plant biomass, oil, meal,animal feed, flour, flakes, bran, lint, hulls, and processed seed.
 22. Amethod of producing a seed, the method comprising: planting at least oneseed comprising a polynucleotide encoding the insect inhibitoryrecombinant polypeptide of claim 1; growing at least one plant from theat least one seed; and harvesting a seed from the at least one plant,wherein the harvested seed comprises the polynucleotide encoding theinsect inhibitory recombinant polypeptide.